EP0772619B2 - Immunomodulatory oligonucleotides - Google Patents

Immunomodulatory oligonucleotides Download PDF

Info

Publication number
EP0772619B2
EP0772619B2 EP95911630A EP95911630A EP0772619B2 EP 0772619 B2 EP0772619 B2 EP 0772619B2 EP 95911630 A EP95911630 A EP 95911630A EP 95911630 A EP95911630 A EP 95911630A EP 0772619 B2 EP0772619 B2 EP 0772619B2
Authority
EP
European Patent Office
Prior art keywords
composition
odn
immunostimulatory oligonucleotide
cells
subject
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP95911630A
Other languages
German (de)
French (fr)
Other versions
EP0772619B1 (en
EP0772619A1 (en
Inventor
Arthur M. Krieg
Dennis Klinman
Alfred D. Steinberg
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
UNITED STATES OF AMERICA, AS REPRESENTED BY THE SE
University of Iowa Research Foundation UIRF
Coley Pharmaceutical Group Inc
Original Assignee
University of Iowa Research Foundation UIRF
US Department of Health and Human Services
Coley Pharmaceutical Group Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=23056334&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=EP0772619(B2) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by University of Iowa Research Foundation UIRF, US Department of Health and Human Services, Coley Pharmaceutical Group Inc filed Critical University of Iowa Research Foundation UIRF
Priority to EP01202814A priority Critical patent/EP1167379A3/en
Priority to DE69535036T priority patent/DE69535036T3/en
Priority to EP01202811A priority patent/EP1167377B2/en
Priority to EP01202813A priority patent/EP1167378B1/en
Publication of EP0772619A1 publication Critical patent/EP0772619A1/en
Application granted granted Critical
Publication of EP0772619B1 publication Critical patent/EP0772619B1/en
Publication of EP0772619B2 publication Critical patent/EP0772619B2/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • DNA binds to cell membrane and is internalized
  • oligodeoxyribonucleotides are able.to enter cells in a saturable, sequence independent, and temperature and energy dependent fashion (reviewed in Jaroszewski, J.W., and J.S. Cohen. 1991.
  • Lymphocyte ODN uptake has been shown to be regulated by cell activation.
  • Spleen cells stimulated with the B cell mitogen LPS had dramatically enhanced ODN uptake in the B cell population, while spleen cells treated with the T cell mitogen Con A showed enhanced ODN uptake by T but not B cells (Krieg, A.M., F. Gmelig-Meyling, M.F. Gourley, WJ. Kisch, L.A. Chrisey, and A.D. Steinberg. 1991. "Uptake of oligodeoxyribonucleotides by lymphoid cells is heterogeneous and inducible”. Antisense Research and Development 1:161).
  • poly (I,C) which is a potent inducer of IFN production as well as a macrophage activator and inducer of NK activity
  • I,C a potent inducer of IFN production as well as a macrophage activator and inducer of NK activity
  • Guanine ribonucleotides substituted at the C8 position with either a bromine or a thiol group are B cell mitogens and may replace "B cell differentiation factors" ( Feldbush, T.L., and Z.K. Ballas. 1985. "Lymphokine-like activity of 8-mercaptoguanosine: induction ofT and B cell differentiation". J. Immunol. 134:3204 ; and Goodman, M.G. 1986. "Mechanism of synergy between T cell signals and C8-substituted guanine nucleosides in humoral immunity: B lymphotropic cytokines induce responsiveness to 8-mercaptoguanosine". J. Immunol. 136:3335 ).
  • 8-mercaptoguanosine and 8-bromoguanosine also can substitute for the cytokine requirement for the generation ofMHC restricted CTL (Feldbush, T.L., 1985. cited supra), augment murine NK activity ( Koo, G.C., M.E. Jewell, C.L. Manyak, N.H. Sigal, and L.S. Wicker. 1988. "Activation of murine natural killer cells and macrophages by 8-bromoguanosine". J. Immunol. 140:3249 ), and synergize with IL-2 in inducing murine LAK generation ( Thompson, R.A., and Z.K. Ballas. 1990. "Lymphokine-activated killer (LAK) cells. V.
  • CREB cAMP response element binding protein
  • ATF activating transcription factor
  • CREB/ATF family of transcription factors is a ubiquitously expressed class of transcription factors of which 11 members have so far been cloned (reviewed in de Groot, R-P., and P. Sassone-Corsi: "Hormonal control of gene expression: Multiplicity and versatility of cyclic adenosine 3',5'-monophosphate-responsive nuclear regulators". Mol. Endocrin. 7:145,1993 ; Lee, K.A.W., and N. Masson: "Transcriptional regulation by CREB and its relatives". Biochim. Biophys. Acta 1174:221,1993 .).
  • bZip basic region/leucine zipper
  • All cells appear to express one or more CREB/ATF proteins, but the members expressed and the regulation of mRNA splicing appear to be tissue-specific. Differential splicing of activation domains can determine whether a particular CREB/ATF protein will be a transcriptional inhibitor or activator. Many CREB/ATF proteins activate viral transcription, but some splicing variants which lack the activation domain are inhibitory.
  • CREB/ATF proteins can bind DNA as homo- or hetero- dimers through the cAMP response element, the CRE, the consensus form of which is the unmethylated sequence TGACGTC (binding is abolished if the CpG is methylated) ( Iguchi-Ariga, S.M.M., and W. Schaffner "CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation". Genes & Develop. 3:612,1989 .).
  • CREB/ATF proteins appear to regulate the expression of multiple genes through the CRE including immunologically important genes such as foa, jun B, Rb-1, IL-6, IL-1 ( Tsukada, J., K. Saito, W.R. Waterman, A.C. Webb, and P.E. Auron: "Transcription factors NF-IL6 and CREB recognize a common essential site in the human prointerleukin 1 ⁇ gene". Mol.
  • Ehrlich "Binding of AP-1/CREB proteins and of MDBP to contiguous sites downstream of the human TGF-B gene". Biochim. Biophys. Acta 1219:55,1994 .), TGF- ⁇ 2, class II MHC ( Cox, P.M., and C.R. Goding: "An ATF/CREB binding motif is required for aberrant constitutive expression of the MHC class II DRa promoter and activation by SV40 T-antigen". Nucl. Acids Res.
  • CREB can also mediate transcriptional responses to changes in intracellular Ca ++ concentration ( Sheng, M., G. McFadden, and M.E. Greenberg: "Membrane depolarization and calcium induce c-fos transcription via phosphorylation of transcription factor CREB". Neuron 4:571, 1990 ).
  • CREB protein kinase A
  • CBP protein kinase A
  • CBP basal transcription factor
  • TFIIB basal transcription factor
  • CREB also has been reportod to interact with dTAFII 110, a TATA binding protein-assodated factor whose binding may regulate transcription ( Ferreri, K., G. Gill, and M.
  • CREB/ATF proteins can specifically bind multiple other nuclear factors ( Hoeffler, J.P., J.W. Lustbader, and C.-Y. Chen: "Identification of multiple nuclear factors that interact with cyclic adenosine 3',5'-monophosphate response element-binding protein and activating transcription factor-2 by protein-protein interactions". Mol. Endocrinol. 5:256, 1991 ) but the biologic significance of most of these interactions is unknown.
  • CREB is normally thought to bind DNA either as a homodimer or as a heterodimer with several other proteins.
  • CREB monomers constitutively activate transcription Krajewski, W., and K.A.W. Lee: "A monomeric derivative of the cellular transcription factor CREB functions as a constitutive activator”. Mol. Cell. Biol. 14:7204,1994 .).
  • cytomegalovirus immediate early promoter one of the strongest known mammalian promoters, contains eleven copies of the CRE which are essential for promoter function ( Chang, Y.-N., S. Crawford, J. Stall, D.R. Rawlins, K.-T. Jeang, and G.S. Hayward: "The palindromic series I repeats in the simian cytomegalovirus major immediate-early promoter behave as both strong basal enhancers and cyclic AMP response elements". J. Virol. 64:264, 1990 ).
  • At least some of the tmnscriptional activating effects of the adenovirus E1A protein, which induces many promoters, are due to its binding to the DNA binding domain of the CREB/ATF protein, ATF-2, which mediates E1A inducible transcription activation ( Liu, F., and M.R. Green: "Promoter targeting by adenovirus Ela through interaction with different cellular DNA-binding domains". Nature 368-520,1994 ). It has also been suggested that E1A binds to the CREB-binding protein, CBP ( Arany, Z., W.R. Sellers, D.M. Livingston, and R.
  • CBP Arany, Z., W.R. Sellers, D.M. Livingston, and R.
  • HTLV-1 Human T lymphotropic virus-I
  • Tax which binds to CREB/ATF proteins and redirects them from their normal cellular binding sites to different DNA sequences (flanked by G- and C-rich sequences) present within the HTLV transcriptional enhancer ( Paca-Uccaralertkun, S., L.-J. Zhao, N. Adya, J.V. Cross. B.R.
  • the instant invention is based on the finding that certain oligonucleotides containing unmethylated cytosine-guanine (CpG) dinucleotides activate lymphoeytes as evidenced by in vitro and in vivo data. Based on this finding, the invention features, in one aspect, novel immunostimulatory oligonucleotide compositions.
  • the present invention provides a composition, for use as a medicament, comprising a vaccine and a synthetic immunostimulatory oligonucleotide of at least 8 bases in size, which contains at least one unmethylated CpG dinucleotide and is an adjuvant for the vaccine.
  • the invention provides the use of a composition in accordance with the first aspect of the invention, for the manufacture of a medicament for vaccinating a subject.
  • the invention provides the use of a synthetic immunostimulatory oligonucleotide of at least 8 bases in size, which contains at least one unmethylated CpG dinucleotide, for the manufacture of a medicament for administration to a subject in conjunction with a vaccine, as an adjuvant for the vaccine.
  • an immunostimulatory oligonucleotide is synthetic, between 8 to 100 bases in size and contains a consensus mitogenic CpG motif represented by the formula: 5' X 1 X 2 CGX 3 X 4 3' wherein C and G are unmethylated, X 1 , X 2 , X 3 and X 4 are nucleotides and a GCG trinuclootide sequence is not present at or near the 5' and 3' termini.
  • CpG containing immunostimulatory oligonucleotides are preferably in the range of 8 to 40 bases in size.
  • Prolonged immunostimulation can be obtained using stabilized oligonucleotides, particularly phosphorothioate stabilized oligonucleotides.
  • Enhanced immunostimulatory activity has been observed where X 1 X 2 is the dinucleotide GpA and/or X 3 X 4 is the dinucleotide is most preferably TpC or also TpT. Further enhanced immunostimulatory activity has been observed where the consensus motif X 1 X 2 CGX 3 X 4 is preceded on the 5' end by a T.
  • lymphocytes can either be obtained from a subject and stimulated ex vivo upon contact with an appropriate oligonucleotide; or a non-methylated CpG containing oligonucleotide can be administered to a subject to facilitate in vivo activation of a subject's lymphocytes.
  • Activated lymphocytes stimulated by the methods described herein (e.g. either ex vivo or in vivo ), can boost a subject's immune response.
  • immunostimulatory oligonucleotides can therefore be used to treat, prevent or ameliorate an immune system deficiency (e.g., a tumor or cancer or a viral, fungal, bacterial or parasitic infection in a subject
  • immunostimulatory oligonucleotides can also be administered as a vaccine adjuvant, to stimulate a subject's response to a vaccine.
  • oligonucleotide or “oligo” shall mean multiple nucleotides (i.e. molecules comprising a sugar (e.g. ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g. cytosine (C), thymine (T) or uracil (U)) or a substituted purine (e.g. adenine (A) or guanine (G)).
  • oligonucleotide'' as used herein refers to both oligoribonucleotides (ORNs) and oligodeoxyribonucleotides (ODNs).
  • oligonucleotide shall also include oligonucleosides (i.e. an oligonucleotide minus the phosphate) and any other organic base containing polymer. Oligonucleotides can be obtained from existing nucleic acid sources (e.g. genomic or cDNA), but are preferably synthetic (e.g. produced by oligonucleotide synthesis).
  • a “stabilized oligonucleotide” shall mean an oligonucleotide that is relatively resistant to in vivo degradation (e.g. via an exo- or endo-nuclease).
  • Preferred stabilized oligonucleotides of the instant invention have a modified phosphate backbone.
  • Especially preferred oligonucleotides have a phosphorothioate modified phosphate backbone (i.e. at least one of the phosphate oxygens is replaced by sulfur).
  • oligonucleotides include: nonionic DNA analogs, such as alkyl- and aryl- phosphonates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), phosphodiester and alkylphosphotriesters, in which the charged oxygen moiety is alkylated. Oligonucleotides which contain a diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini have also been shown to be substantially resistant to nuclease degradation.
  • an “immunostimulatory oligonucleotide”, “immunostimulatory CpG containing oligonucleotide”, or “CpG ODN” refer to an oligonucleotide, which contains a cytosine, guanine dinucleotide sequence and stimulates (e.g. has a mitogenic effect) on vertebrate lymphocytes.
  • Preferred immunostimulatory oligonucleotides are between 8 to 100 bases in size and contain a consensus mitogenic CpG motif represented by the formula: 5' X 1 X 2 CGX 3 X 4 3' wherein C and G are unmethylated, X 1 , X 2 , X 3 and X 4 are nucleotides and a GCG trinucleotide sequence is not present at or near the 5' and 3' termini.
  • the immunostimulatory oligonucleotides range between 8 to 40 bases in size.
  • the immunostimulatory oligonucleotides are preferably stabilized oligonucleotides, particularly preferred are phosphorothioate stabilized oligonucleotides.
  • X 1 X 2 is the dinucleotide GpA.
  • X 3 X 4 is preferably the dinucleotide TpC or also TpT.
  • the consensus motif X 1 X 2 CGX 3 X 4 is preceded on the 5' end by a T. Particularly preferred consensus sequences are TGACGTT or TGACGTC.
  • Prolonged immunostimulation can be obtained using stabilized oligonucleotides, particularly phosphorothioate stabilized.
  • “Palindromic sequence” shall mean an inverted repeat (i.e. a sequence such as ABCDEE'D'CB'A' in which A and A' are bases capable of forming the usual Watson-Crick base pairs. In vivo, such sequences may form double stranded structures.
  • oligonucleotide delivery complex shall mean an oligonucleotide associated with (e.g. ionically or covalently bound to; or encapsulated within) a targeting means (e.g. a molecule that results in higher affinity binding to target cell (e.g. B-cell and natural killer (NK) cell) surfaces and/or increased cellular uptake by target cells).
  • a targeting means e.g. a molecule that results in higher affinity binding to target cell (e.g. B-cell and natural killer (NK) cell) surfaces and/or increased cellular uptake by target cells.
  • oligonucleotide delivery complexes include oligonucleotides associated with: a sterol (e.g. cholesterol), a lipid (e.g. a cationic lipid, virosome or liposome), or a target cell specific binding agent (e.g. a ligand recognized by target cell specific receptor).
  • Preferred complexes must be sufficiently stable in vivo to prevent significant uncoupling prior to internalization by the target cell.
  • the complex should be cleavable under appropriate conditions within the cell so that the oligonucleotide is released in a functional form.
  • an "immune system deficiency” shall mean a disease or disorder in which the subject's immune system is not functioning in normal capacity or in which it would be useful to boost a subject's immune response for example to eliminate a tumor or cancer (e.g. tumors of the brain, lung (e.g. small cell and non-small cell), ovary, breast, prostate, colon, as well as other carcinomas and sarcomas) or a viral (e.g. HIV, herpes), fungal (e.g. Candida sp.), bacterial or parasitic (e.g. Leishmania, Toxoplasma) infection in a subject.
  • a tumor or cancer e.g. tumors of the brain, lung (e.g. small cell and non-small cell), ovary, breast, prostate, colon, as well as other carcinomas and sarcomas) or a viral (e.g. HIV, herpes), fungal (e.g. Candida sp.), bacterial or parasitic (e.g.
  • a “disease associated with immune system activation” shall mean a disease or condition caused or exacerbated by activation of the subject's immune system. Examples include systemic lupus erythematosus, sepsis and autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.
  • a "subject” shall mean a human or vertebrate animal including a dog, cat, horse, cow, pig, sheep, goat, chicken, monkey, rat, mouse, etc.
  • ODN 1 and 2 were synthesized. These ODNs, including the two original “controls” (ODN 1 and 2) and two originally synthesized as "antisense” (ODN 3D and 3M; Krieg, A.M. J. Immunol. 143:2448 (1989 )), were then examined for in vitro effects on spleen cells (representative sequences are listed in Table 1).
  • ODN that contained CpG dinucleotides induced B cell activation and IgM secretion; the magnitude of this stimulation typically could be increased by adding more CpG dinucleotides (Table 1; compare ODN 2 to 2a or 3D to 3Da and 3Db). Stimulation did not appear to result from an antisense mechanism or impurity. ODN caused no detectable activation of ⁇ or other T cell populations.
  • the optimal stimulatory motif was determined to consist of a CpG flanked by two 5' purines (preferably a GpA dinucleotide) and two 3' pyrimidines (preferably a TpT or TpC dinucleotide). Mutations of ODN to bring the CpG motif closer to this ideal improved stimulation (e.g. compare ODN 2 to 2e; 3M to 3Md) while mutations that disturbed the motif reduced stimulation (e.g. compare ODN 3D to 3Df; 4 to 4b, 4c and 4d). On the other hand, mutations outside the CpG motif did not reduce stimulation (e.g. compare ODN 1 to 1d; 3D to 3Dg; 3M to 3Me).
  • ODNs shorter than 8 bases were non-stimulatory (e.g. ODN 4e).
  • ODN 4e the most stimulatory sequence identified was TCAACGTT (ODN 4) which contains the self complementary "palindrome" AACGTT.
  • ODN containing Gs at both ends showed increased stimulation, particularly if the the ODN were rendered nuclease resistant by phosphorothioate modification of the terminal intemucleotide linkages.
  • ODN 1585 (5' GGGGTCAACGTTCAGGGGGG 3' (SEQ ID NO:1)), in which the first two and last five internucleotide linkages are phosphorothioate modified caused an average 25.4 fold increase in mouse spleen cell proliferation compared to an average 3.2 fold increase in proliferation induced by ODN 1638, which has the same sequence as ODN 1585 except that the 10 Gs at the two ends are replaced by 10 As.
  • the effect of the G-rich ends is cis; addition of an ODN with poly G ends but no CpG motif to cells along with 1638 gave no increased proliferation.
  • ODN 4b,4c octamer ODN containing a 6 base palindrome with a TpC dinucleotide at the 5' end were also active if they were dose to the optimal motif (e.g. ODN 4b,4c). Other dinucleotides at the 5' end gave reduced stimulation (eg ODN 4f; all sixteen possible dinucleotides were tested). The presence of a 3' dinucleotide was insufficient to compensate for the lack of a 5' dinucleotide (eg. ODN 4g). Disruption of the palindrome eliminated stimulation in octamer ODN (eg., ODN 4h), but palindromes were not required in longer ODN.
  • Stimulation indexes are the means and std. dev. derived from at least 3 separate experiments, and are compared to wells cultured with no added ODN. ND - not done. CpG dinucleotides are underlined. Dots indicate identity; dashes indicate deletions. Z indicates 5 methyl cytosine.)
  • CpG-ODN induced cycling in more than 95% of B cells (Table 2).
  • Splenic B lymphocytes sorted by flow cytometry into CD23- (marginal zone) and CD23+ (follicular) subpopulations were equally responsive to ODN- induced stimulation, as were both resting and activated populations ofB cells isolated by fractionation over Percoll gradients.
  • CpG ODN peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • CLL chronic lymphocytic leukemia
  • Control ODN containing no CpG dinucleotide sequence showed no effect on the basal proliferation of 442 cpm and 874 cpm (proliferation measured by 3 H thymidine incorporation) of the human cells.
  • a phosphorothioate modified CpG ODN 3Md SEQ m NO: 25
  • a phosphorothioate modified CpG ODN 3Md induced increased proliferation of 7210 and 86795 cpm respectively in the two patients at a concentration of just 1 ⁇ M. Since these cells had been frozen, they may have been less responsive to the oligos than fresh cells in vivo
  • cells from CLL patients typically are non-proliferating, which is why traditional chemotherapy is not effective.
  • Certain B cell lines such as WEHI-231 are induced to undergo growth arrest and/or apoptosis in response to crosslinking of their antigen receptor by anti-IgM ( Jakway, J.P. et al., "Growth regulation of the B lymphoma cell line WEHI-231 by anti-immunoglobulin, lipopolysaccharide and other bacterial products" J. Imminol. 137: 2225 (1986 ); Tsubata, T., J. Wu and T. Honjo: B-cell apoptosis induced by antigen receptor crosslinking is blocked by a T-cell signal through CD40.” Nature 364: 645 (1993 )).
  • WEHI-231 cells are rescued from this growth arrest by certain stimuli such as LPS and by the CD40 ligand. ODN containing the CpG motif were also found to protect WEHI-231 from anti-IgM induced growth arrest, indicating that accessory cell populations are not required for the effect.
  • CpG ODN cytokines and prostaglandins in vitro and in vivo were measured. Unlike LPS, CpG ODN were not found to induce purified macrophages to produce prostaglandin PGE2. In fact, no apparent direct effect of CpG ODN was detected on either macrophages or T cells. In vivo or in whole spleen cells, no significant increase in the following interleukins: IL-2. IL-3, IL-4, or IL-10 was detected within the first six hours. However, the level of IL-6 increased strikingly within 2 hours in the serum of mice injected with CpG ODN. Increased expression of IL-12 and interferon gamma (EFN- ⁇ ) by spleen cells was also detected within the first two hours.
  • EFN- ⁇ interferon gamma
  • mice were injected once intraperitoneally with PBS or phosphorothioate CpG or non-CpG ODN at a dose of 33 mg/kg (approximately 500 ⁇ g/mouse).
  • Pharmacokinetic studies in mice indicate that this dose of phosphorothioate gives levels of approximately 10 ⁇ g/g in spleen tissue (within the effective concentration range determined from the in vitro studies described herein) for longer than twenty-four hours ( Agrawal, S. et al. (1991) Proc. Natl. Acad Sci. USA 91:7595 ).
  • Spleen cells from mice were examined twenty-four hours after ODN injection for expression ofB cells surface activation markers Ly-6A/E, Bla-1, and class II MHC using three color flow cytometry and for their spontaneous proliferation using 3 H thymidine. Expression of all three activation markers was significantly increased in B cells from mice injected with CpG ODN, but not from mice injected with PBS or non-CpG ODN. Spontaneous 3 H thymidine incorporation was increased by 2-6 fold in spleen cells from mice injected with the stimulatory ODN compared to PBS or non-CpG ODN-injected mice. After 4 days, serum IgM levels in mice injected with CpG ODN in vivo were increased by approximately 3-fold compared to controls. Consistent with the inability of these agents to activate T cells, there was minimal change in T cell expression of the IL-2R or CD-44.
  • Example 4 experiments were conducted to determine whether CpG containing oligonucleotides stimulated the activity of natural killer (NK) cells in addition to B cells. As shown in Table 3, a marked induction ofNK activity among spleen cells cultured with CpG ODN 1 and 3Dd was observed. In contrast, there was relatively no induction in effectors that had been treated with non-CpG control ODN.
  • NK natural killer
  • Table 3 Induction Of NK Activity By CpG Oligodeoxynucleotides (ODN) % YAC-1 Specific Lysis* % 2C11 Specific Lysis Effector: Target Effector: Target ODN 50:1 100:1 50:1 100:1 None -1.1 -1.4 15.3 16.6 1 16.1 24.5 38.7 47.2 3Dd 17.1 27.0 37.0 40.0 non-CpG ODN -1.6 -1.7 14.8 15.4
  • ODN containing CpG dinucleotides that are not in the stimulatory motif described above were found to block the stimulatory effect of other mitogenic CpG ODN.
  • an atypical CpG motif consisting of a GCG near or at the 5' and/or 3' end of CpG ODN actually inhibited stimulation of proliferation by other CpG motifs.
  • Methylation or substitution of the cytosine in a GCG motif reverses this effect.
  • a GCG motif in an ODN has a modest mitogenic effect, though far lower than that seen with the preferred CpG motif.
  • CpG-ODN did not induce any detectable Ca 2+ flux, changes in protein tyrosine phosphorylation, or IP 3 generation.
  • Flow cytometry with FITC-conjugated ODN with or without a CpG motif was performed as described in Zhao, Q et al.,(Antisense Research and Development 3:53-66 (1993) ), and showed equivalent membrane binding, cellular uptake, efflux, and intracellular localization. This suggests that there may not be cell membrane proteins specific for CpG ODN.
  • the optimal CpG motif (TGACGTT/C is identical to the CRE (cyclic AMP response element). Like the mitogenic effects of CpG ODN, binding of CREB to the CRE is abolished if the central CpG is methylated. Electrophoretic mobility shift assays were used to determine whether CpG ODN, which are single stranded, could compete with the binding of B cell CREB/ATF proteins to their normal binding site, the doublestranded CRE. Competition assays demonstrated that single stranded ODN containing CpG motifs could completely compete the binding of CREB to its binding site, while ODN without CpG motifs could not.
  • the stimulatory CpG motif is common in microbial genomic DNA, but quite rare in vertebrate DNA.
  • bacterial DNA has been reported to induce B cell proliferation and immunoglobulin (Ig) production, while mammalian DNA does not (Messina, J.P. et al., J Immunol. 147:1759 (1991 )).
  • Ig immunoglobulin
  • Example 3 in which methymon of bacterial DNA with CpG methylase was found to abolish mitogenicity, demonstrates that the difference in CpG status is the cause of B cell stimulation by bacterial DNA. This data supports the following conclusion: that umnethylated CpG dinucleotides present within bacterial DNA are responsible for the stimulatory effects of bacterial DNA.
  • lymphocyte activation by the CpG motif represents an immune defense mechanism that can thereby distinguish-bacterial from host DNA.
  • Host DNA would induce little or no lymphocyte activation due to it CpG suppression and methylation.
  • Bacterial DNA would cause selective lymphocyte activation in infected tissues. Since the CpG pathway synergizes with B cell activation through the antigen receptor, B cells bearing antigen receptor specific for bacterial antigens would receive one activation signal through cell membrane Ig and a second signal from bacterial DNA, and would therefore tend to be preferentially activated. The interrelationship of this pathway with other pathways of B cell activation provide a physiologic mechanism employing a polyclonal antigen to induce antigen-specific responses.
  • oligonucleotides can be synthesized de novo using any of a number of procedures well known in the art.
  • the ⁇ -cyanoethyl phosphoramidite method S.L. Beaucage and M.H. Caruthers, (1981) Tet. Let. 22:1859
  • nucleoside H-phosphonate method Garegg et al., (1986) Tet. Let. 27: 4051-4054 ; Froehler et al., (1986) Nucl. Acid Res. 14: 5399-5407 ; Garegg et al., (1986) Tet. Let.
  • oligonucleotides can be prepared from existing nucleic acid sequences (e.g. genomic or cDNA) using known techniques, such as those employing restriction enzymes, exonucleases or endonucleases.
  • oligonucleotides are preferably relatively resistant to degradation (e.g. via endo- and exo- nucleases). Oligonucleotide stabilization can be accomplished via phosphate backbone modifications. A preferred stabilized oligonucleotide has a phosphorothioate modified backbone. The pharmacokinetics of phosphorothioate ODN show that they have a systemic half-life of forty-eight hours in rodents and suggest that they may be useful for in vivo applications ( Agrawal, S. et al. (1991) Proc. Natl. Acad Sci. USA 88:7595 ).
  • Phosphorothioates may be synthesized using automated techniques employing either phosphoramidate or H phosphonate chemistries.
  • Aryl- and alkyl- phosphonates can be made e.g. (as described in U.S. Patent No. 4,469,863 ); and alkylphosphotriesters (in which the charged oxygen moiety is alkylated as described in U.S. Patent No. 5,023,243 and European Patent No. 092,574 ) can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other DNA backbone modifications and substitutions have been described ( Uhlmann, E. and Peyman, A. (1990) Chem Rev. 90:544 ; Goodchild, J. (1990) Bioconjugate Chem. 1:165 ).
  • oligonucleotides may be associated with a molecule that results in higher affinity binding to target cell (e.g. B-cell and natural killer (NK) cell) surfaces and/or increased cellular uptake by target cells to form an "oligonucleotide delivery complex".
  • Oligonucleotides can be ionically, or covalently associated with appropriate molecules using techniques which are well known in the art.
  • a variety of coupling or crosslinking agents can be used e.g. protein A, carbodiimide, and N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP).
  • Oligonucleotides can alternatively be encapsulated in liposomes or virosomes using well-known techniques.
  • oligonucleotides containing at least one unmethylated CpG dinucleotide can be administered to a subject in vivo to treat an "immune system deficiency".
  • oligonucleotides containing at least one unmethylated CpG dinucleotide can be contacted with lymphocytes (e.g. B cells or NK cells) obtained from a subject having an immune system deficiency ex vivo and activated lymphocytes can then be reimplanted in the subject.
  • lymphocytes e.g. B cells or NK cells
  • Immunostimulatory oligonucleotides can be administered to a subject in conjunction with the vaccine, as an adjuvant, to boost a subject's immune system to effect better response from the vaccine, slightly before or at the same time as the vaccine.
  • CpG ODN also increased natural killer cell activity in both human and murine cells. Induction of NK activity may likewise be beneficial in cancer immunotherapy.
  • an effective amount of an appropriate oligonucleotide alone or formulated as an oligonucleotide delivery complex can be administered to a subject by any mode allowing the oligonucleotide to be taken up by the appropriate target cells (e.g. B-cells and NK cells).
  • Preferred routes of administration include oral and transdermal (e.g. via a patch).
  • Other routes of administration include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, etc.). The injection can be in a bolus or a continuous infusion.
  • an oligonucleotide alone or as an oligonucleotide delivery complex can be administered in conjunction with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include substances that can be coadministered with an oligonucleotide or an oligonucleotide delivery complex and allows the oligonucleotide to perform its intended function.
  • examples of such carriers include solutions, solvents, dispersion media, delay agents, emulsions and the like. The use of such media for pharmaceutically active substances are well known in the art. Any other conventional carrier suitable for use with the oligonucleotides falls within the scope of the instant invention.
  • an effective amount of an oligonucleotide refers to that amount necessary or sufficient to realize a desired biologic effect.
  • an effective amount of an oligonucleotide containing at least one unmethylated CpG for treating an immune system deficiency could be that amount necessary to eliminate a tumor, cancer, or bacterial, viral or fungal infection.
  • An effective amount for use as a vaccine adjuvant could be that amount useful for boosting a subject's immune response to a vaccine.
  • the effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular oligonucleotide being administered, the size of the subject, or the severity of the disease or condition.
  • One of ordinary skill in the art can empirically determine the effective amount of a particular oligonucleotide without necessitating undue experimentation.
  • lymphocytes e.g. B cells and NK cells.
  • lymphocytes e.g. B cells and NK cells.
  • these results suggest that the underrepresentation of CpG dinucleotides in animal genomes, and the extensive methylation of cytosines present in such may be explained by the existence of an immune defense mechanism that can distinguish bacterial from host DNA.
  • Host DNA would commonly be present in many anatomic regions and areas of inflammation due to apoptosis (cell death), but generally induces little or no lymphocyte activation.
  • oligonucleotides containing GCG trinucleotide sequences at or near both termini have antiviral activity, independent of any antisense effect due to complementarity between the oligonucleotide and the viral sequence being targeted. Based on this activity, an effective amount of inhibitory oligonucleotides can be administered to a subject to treat or prevent a viral infection.
  • B cells were purified from spleens obtained from 6-12 wk old specific pathogen free DBA/2 or BXSB mice (bred in the University of Iowa animal care facility; no substantial strain differences were noted) that were depleted ofT cells with anti-Thy-1.2 and complement and centrifugation over lympholyte M (Cedarlane Laboratories, Hornby, Ontario, Canada) ("B cells"). B cells contained fewer than 1% CD4 + or CD8 + cells.
  • 8x10 4 B cells were dispensed in triplicate into 96 well microtiter plates in 100 ⁇ l RPMI containing 10% FBS (heat inactivated to 65°C for 30 min.), 50 ⁇ M 2-mercaptoethanol, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, and 2 mM L-glutamate.
  • 20 ⁇ M ODN were added at the start of culture for 20 h at 37°C, cells pulsed with 1 ⁇ Ci of 3 H uridine, and harvested and counted 4 hr later. Ig secreting B cells were enumerated using the ELISA spot assay after culture of whole spleen cells with ODN at 20 ⁇ M for 48 hr.
  • PBMCs perpheral blood monocyte cells
  • CLL chronic lymphocytic leukemia
  • the number ofB cells actively secreting IgM was maximal at this time point, as determined by ELIspot assay ( Klinman, D.M. et al. J. Immunol. 144:506 (1990 )).
  • B cells were incubated for 6 hrs on anti-Ig coated microtiter plates.
  • the Ig they produced (>99% IgM) was detected using phosphatase-labelled anti-Ig (Southern Biotechnology Associated, Birmingham, AL).
  • the antibodies produced by individual B cells were visualized by addition of BCIP (Sigma Chemical Co., St. Louis MO) which forms an insoluble blue precipitate in the presence of phosphatese.
  • the dilution of cells producing 20 - 40 spots/well was used to determine the total number of antibody-secreting B cells/sample. All assays were performed in triplicate. In some experiments, culture supernatants were assayed for IgM by ELISA, and showed similar increases in response to CpG-ODN.
  • Example 3 B cell Stimulation by Bacterial DNA
  • DBA/2 B cells were cultured with no DNA or 50 ⁇ g/ml of a) Micrococcus lysodeikticus; b) NZB/N mouse spleen; and c) NFS/N mouse spleen genomic DNAs for 48 hours, then pulsed with 3 H thymidine for 4 hours prior to cell harvest.
  • Duplicate DNA samples were digested with DNAse I for 30 minutes at 37 C prior to addition to cell cultures.
  • E coli DNA also induced an 8.8 fold increase in the number of IgM secreting B cells by 48 hours using the ELISA-spot assay.
  • DBA/2 B cells were cultured with either no additive, 50 ⁇ g/ml LPS or the ODN 1; 1a; 4; or 4a at 20 uM. Cells were cultured and harvested at 4, 8, 24 and 48 hours. BXSB cells were cultured as in Example 1 with 5, 10, 20,40 or 80 ⁇ M of ODN 1; 1a; 4; or 4a or LPS. In this experiment, wells with no ODN had 3833 cpm. Each experiment was performed at least three times with similar results. Standard deviations of the triplicate wells were ⁇ 5%.
  • 10 x 10 6 C57BL/6 spleen cells were cultured in two ml RPMI (supplemented as described for Example 1) with or without 40 ⁇ M CpG or non-CpG ODN for forty-eight hours. Cells were washed, and then used as effector cells in a short term 51 Cr release assay with YAC-1 and 2C11, two NK sensitive target cell lines (Ballas, Z. K. et al. (1993) J . Immunol. 150:17). Effector cells were added at various concentrations to 10 4 51 Cr-labeled target cells in V-bottom microtiter plates in 0.2 ml, and incubated in 5% CO 2 for 4 hr. at 37°C.
  • Percent specific lysis was determined by calculating the ratio of the 51 Cr released in the presence of effector cells minus the 51 Cr released when the target cells are cultured alone, over the total counts released after cell lysis in 2% acetic acid minus the 51 Cr cpm released when the cells are cultured alone.
  • mice were weighed and injected IP with 0.25 ml of sterile PBS or the indicated phophomthioate ODN dissolved in PBS. Twenty four hours later, spleen cells were harvested, washed, and stained for flow cytometry using phycoerythrin conjugated 6B2 to gate on B cells in conjunction with biotin conjugated anti Ly-6A/E or anti-Ia d (Pharmingen, San Diego, CA) or anti-Bla-1 ( Hardy, R.R. et al., J. Exp. Med. 159:1169 (1984 ). Two mice were studied for each condition and analyzed individually.
  • B cells were cultured with phosphorothioate ODN with the sequence of control ODN 1a or the CpG ODN 1d and 3Db and then either pulsed after 20 hr with 3 H uridine or after 44 hr with 3 H thymidine before harvesting and determining cpm.
  • WEHI-231 cells (5 x 10 4 /well) were cultured for 1 hr. at 37 C in the presence or absence ofLPS or the control ODN 1a or the CpG ODN 1d and 3Db before addition of anti-IgM (1 ⁇ /ml). Cells were cultured for a further 20 hr. before a 4 hr. pulse with 2 ⁇ Ci/well 3 H thymidine. In this experiment, cells with no ODN or anti-IgM gave 90.4 x 10 3 by addition of anti-IgM.
  • the phosphodiester ODN shown in Table 1 gave similar protection, though with some nonspecific suppression due to ODN degradation. Each experiment was repeated at least 3 times with similar results.
  • mice DBA/2 female mice (2 mos. old) were injected IP with 500 ⁇ g CpG or control phosphorothioate ODN. At various time points after injection, the mice were bled. Two mice were studied for each time point. IL-6 was measured by Elisa, and IL-6 concentration was calculated by comparison to a standard curve generated using recombinant EL-6. The sensitivity of the assay was 10 pg/ml. Levels were undetectable after 8 hr.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Oncology (AREA)
  • General Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Communicable Diseases (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Mycology (AREA)
  • Neurology (AREA)
  • Analytical Chemistry (AREA)
  • Plant Pathology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Dermatology (AREA)
  • Neurosurgery (AREA)
  • Rheumatology (AREA)

Abstract

Stabilised immunostimulatory oligonucleotides which contain at least one unmethylated CpG dinucleotide for medicinal use.

Description

    Background of the Invention DNA binds to cell membrane and is internalized
  • In the 1970's, several investigators reported the binding of high molecular weight DNA to cell membranes (Lerner, R.A., W. Meinke, and D.A. Goldstein. 1971. "Membrane-associated DNA in the cytoplasm of diploid human lymphocytes". Proc. Natl. Acad Sci. USA 68:1212; Agrawal, S.K., R.W. Wagner, P.K. McAllister, and B. Rosenberg. 1975. "Cell-surface-associated nucleic acid in tumorigenic cells made visible with platinum-pyrimidine complexes by electron microscopy". Proc. Natl. Acad. Sci. USA 72:928). In 1985 Bennett et al. presented the first evidence that DNA binding to lymphocytes is similar to a ligand receptor interaction: binding is saturable, competitive, and leads to DNA endocytosis and degradation (Bennett, R.M., G.T. Gabor, and M.M. Merritt. 1985. "DNA binding to human leukocytes. Evidence for a receptor-mediated association, internalization, and degradation of DNA". J. Clin. Invest. 76:2182). Like DNA, oligodeoxyribonucleotides (ODNs) are able.to enter cells in a saturable, sequence independent, and temperature and energy dependent fashion (reviewed in Jaroszewski, J.W., and J.S. Cohen. 1991. "Cellular uptake of antisense oligodeoxynucleotides". Advanced Drug Delivery Reviews 6:235; Akhtar, S., Y. Shoji, and R.L. Juliano. 1992. "Pharmaceutical aspects of the biological stability and membrane transport characteristics of antisense oligonucleotides". In: Gene Regulation: Biology of Antisense RNA and DNA. R.P. Erickson, and J.G. Izant, eds. Raven Press, Ltd. New York, pp. 133; and Zhao, Q., T. Waldschmidt, E. Fisher, C.J. Herrera, and A.M. Krieg., 1994. "Stage specific oligonucleotide uptake in murine bone marrow B cell precursors". Blood. 84:3660). No receptor for DNA or ODN uptake has yet been cloned, and it is not yet clear whether ODN binding and cell uptake occurs through the same or a different mechanism from that of high molecular weight DNA.
  • Lymphocyte ODN uptake has been shown to be regulated by cell activation. Spleen cells stimulated with the B cell mitogen LPS had dramatically enhanced ODN uptake in the B cell population, while spleen cells treated with the T cell mitogen Con A showed enhanced ODN uptake by T but not B cells (Krieg, A.M., F. Gmelig-Meyling, M.F. Gourley, WJ. Kisch, L.A. Chrisey, and A.D. Steinberg. 1991. "Uptake of oligodeoxyribonucleotides by lymphoid cells is heterogeneous and inducible". Antisense Research and Development 1:161).
    • Immune effects of nucleic acids
  • Several polynucleotides have been extensively evaluated as biological response modifiers. Perhaps the best example is poly (I,C) which is a potent inducer of IFN production as well as a macrophage activator and inducer of NK activity (Talmadge, J.E., J. Adams, H. Phillips, M. Collins, B. Lenz, M. Schneider, E. Schlick, R. Ruffmann, R.H. Wiltrout, and M.A. Chirigos. 1985. "Immunomodulatory effects in mice of polyinosinic-polycytidylic acid complexed with poly-L:-lysine and carboxymethylcellulose". Cancer Res. 45:1058; Wiltrout, R.H., R.R. Salup, T.A. Twilley, and J.E. Talmadge. 1985. "Immunomodulation of natural killer activity by polyribonucleotides". J. Biol. Resp. Mod 4:512; Krown, S.E. 1986. "Interferons and interferon inducers in cancer treatment". Sem. Oncol. 13:207; and Ewel, C.H., SJ. Urba, W.C. Kopp, J.W. Smith II, R.G. Steis, J.L. Rossio, D.L. Longo, MJ. Jones, W.G. Alvord, C.M. Pinsky, J.M. Beveridge, K.L. McNitt, and S.P. Creekmore. 1992. "Polyinosinic-polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose in combination with interleukin 2 in patients with cancer: clinical and immunological effects". Canc. Res. 52:3005). It appears that this murine NK activation may be due solely to induction of IFN β secretion (Ishikawa, R., and C.A. Biron. 1993. "IFN induction and associated changes in splenic leukocyte distribution". J. Immunol. 150:3713). This activation was specific for the ribose sugar since deoxyribose was ineffective. Its potent in vitro antitumor activity led to several clinical trials using poly (I,C) complexed with poly-L-lysine and carboxymethylcellulose (to reduce degradation by RNAse) (Talmadge, J.E., et al., 1985. cited supra; Wiltrout, RH., et al., 1985. cited supra); Krown, S.E., 1986. cited supra); and Ewel, C.H., et al., 1992. cited supra). Unfortunately, toxic side effects have thus far prevented poly (I,C) from becoming a useful therapeutic agent.
  • Guanine ribonucleotides substituted at the C8 position with either a bromine or a thiol group are B cell mitogens and may replace "B cell differentiation factors" (Feldbush, T.L., and Z.K. Ballas. 1985. "Lymphokine-like activity of 8-mercaptoguanosine: induction ofT and B cell differentiation". J. Immunol. 134:3204; and Goodman, M.G. 1986. "Mechanism of synergy between T cell signals and C8-substituted guanine nucleosides in humoral immunity: B lymphotropic cytokines induce responsiveness to 8-mercaptoguanosine". J. Immunol. 136:3335). 8-mercaptoguanosine and 8-bromoguanosine also can substitute for the cytokine requirement for the generation ofMHC restricted CTL (Feldbush, T.L., 1985. cited supra), augment murine NK activity (Koo, G.C., M.E. Jewell, C.L. Manyak, N.H. Sigal, and L.S. Wicker. 1988. "Activation of murine natural killer cells and macrophages by 8-bromoguanosine". J. Immunol. 140:3249), and synergize with IL-2 in inducing murine LAK generation (Thompson, R.A., and Z.K. Ballas. 1990. "Lymphokine-activated killer (LAK) cells. V. 8-Mercaptoguanosine as an IL-2-sparing agent in LAK generation". J. Immunol. 145:3524). The NK and LAK augmenting activities of these C8-substituted guanosines appear to be due to their induction of IFN (Thompson, R.A., et al. 1990. cited supra). Recently, a 5' triphosphorylated thymidine produced by a mycobacterium was found to be mitogenic for a subset of human γδ T cells (Constant, P., F. Davodeau, M.-A. Peyrat, Y. Poquet, G. Puzo, M. Bonneville, and J.-J. Fournie. 1994. "Stimulation of human γδ T cells by nonpeptidic mycobacterial ligands" Science 264:267). This report indicated the possibility that the immune system may have evolved ways to preferentially respond to microbial nucleic acids.
  • Several observations suggest that certain DNA structures may also have the potential to activate lymphocytes. For example, Bell et al. reported that nucleosomal protein-DNA complexes (but not naked DNA) in spleen cell supernatants caused B cell proliferation and immunoglobulin secretion (Bell, D.A., B. Morrison, and P. VandenBygaart. 1990. "Immunogenic DNA-related factors". J. Clin Invest. 85:1487). In other cases, naked DNA has been reported to have immune effects. For example, Messina et al. have recently reported that 260 to 800 bp fragments of poly (dG)•(dC) and poly (dG•dC) were mitogenic for B cells (Messina, J.P., G.S. Gilkeson, and D.S. Pisetsky. 1993. "The influence of DNA structure on the in vitro stimulation of murine lymphocytes by natural and synthetic polynucleotide antigens". Cell. Immunol. 147:148). Tokunaga, et al. have reported that dG• dC induces γ-IFN and NK activity (Tokumaga, S. Yamamoto, and K. Namba. 1988. "A synthetic single-stranded DNA, poly(dG,dC), induces interferon-α/β and -γ, augments natural killer activity, and suppresses tumor growth" Jpn. J. Cancer Res. 79:682). Aside from such artificial homopolymer sequences, Pisetsky et al. reported that pure mammalian DNA has no detectable immune effects, but that DNA from certain bacteria induces B cell activation and immunoglobulin secretion (Messina, J.P., G.S. Gilkeson, and D.S. Pisetsky. 1991. "Stimulation of in vitro murine lymphocyte proliferation by bacterial DNA". J. Immunol. 147:1759). Assuming that these data did not result from some unusual contaminant, these studies suggested that a particular structure or other characteristic of bacterial DNA renders it capable of triggering B cell activation. Investigations of mycobacterial DNA sequences have demonstrated that ODN which contain certain palindrome sequences can activate NK cells (Yamamoto, S., T. Yamamoto, T. Kataoka, E. Kuramoto, O. Yano, and T. Tokunaga. 1992. "Unique palindromic sequences in synthetic oligonucleotides are required to induce INF and augment INF-mediated natural killer activity". J. Immunol. 148:4072; Kuramoto, E., O. Yano, Y. Kimura, M. Baba, T. Makino, S. Yamamoto, T. Yamamoto, T. Kataoka, and T. Tokunaga. 1992. "Oligonucleotide sequences required for natural killer cell activation". Jpn. J. Cancer Res. 83:1128; and EPO 468 520 A2 ).
  • Several phosphorothioate modified ODN have been reported to induce in vitro or in vivo B cell stimulation (Tanaka, T., C.C. Chu, and W.E. Paul. 1992. "An antisense oligonucleotide complementary to a sequence in Iγ2b increases γ2b germline transcripts, stimulates B cell DNA synthesis, and inhibits immunoglobulin secretion". J. Exp. Med. 175:597; Branda, R.F., A.L. Moore, L. Mathews, J.J. McCormack, and G. Zon. 1993. "Immune stimulation by an antisense oligomer complementary to the rev gene of HIV-1". Biochem. Pharmacol. 45:2037; McIntyre, K.W., K. Lombard-Gillooly, J.R. Perez, C. Kunsch, U.M. Sarmiento, J.D. Larigan, K.T. Landreth, and R. Narayanan. 1993. "A sense phosphorothioate oligonucleotide directed to the initiation codon of transcription factor NF-κ β T65 causes sequence-specific immune stimulation". Antisense Res. Develop. 3:309; and Pisetsky, D.S., and C.F. Reich. 1993. "Stimulation of murine lymphocyte proliferation by a phosphorothioate oligonucleotide with antisense activity for herpes simplex virus". Life Sciences 54:101). These reports do not suggest a common structural motif or sequence element in these ODN that might explain their effects.
    • The CREB/ATF family of transcription factors and their role in replication
  • The cAMP response element binding protein (CREB) and activating transcription factor (ATF) or CREB/ATF family of transcription factors is a ubiquitously expressed class of transcription factors of which 11 members have so far been cloned (reviewed in de Groot, R-P., and P. Sassone-Corsi: "Hormonal control of gene expression: Multiplicity and versatility of cyclic adenosine 3',5'-monophosphate-responsive nuclear regulators". Mol. Endocrin. 7:145,1993; Lee, K.A.W., and N. Masson: "Transcriptional regulation by CREB and its relatives". Biochim. Biophys. Acta 1174:221,1993.). They all belong to the basic region/leucine zipper (bZip) class of proteins. All cells appear to express one or more CREB/ATF proteins, but the members expressed and the regulation of mRNA splicing appear to be tissue-specific. Differential splicing of activation domains can determine whether a particular CREB/ATF protein will be a transcriptional inhibitor or activator. Many CREB/ATF proteins activate viral transcription, but some splicing variants which lack the activation domain are inhibitory. CREB/ATF proteins can bind DNA as homo- or hetero- dimers through the cAMP response element, the CRE, the consensus form of which is the unmethylated sequence TGACGTC (binding is abolished if the CpG is methylated) (Iguchi-Ariga, S.M.M., and W. Schaffner "CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation". Genes & Develop. 3:612,1989.).
  • The transcriptional activity of the CRE is increased during B cell activation (Xie. H. T.C. Chiles, and T.L. Rothstein: "Induction of CREB activity via the surface Ig receptor of B cells". J. Immunol. 151:880, 1993.). CREB/ATF proteins appear to regulate the expression of multiple genes through the CRE including immunologically important genes such as foa, jun B, Rb-1, IL-6, IL-1 (Tsukada, J., K. Saito, W.R. Waterman, A.C. Webb, and P.E. Auron: "Transcription factors NF-IL6 and CREB recognize a common essential site in the human prointerleukin 1β gene". Mol. Cell. Biol. 14:7285, 1994; Gray, G.D.. O.M. Hernandez, D. Hebel, M. Root, J.M. Pow-Sang, and E. Wickstrom: "Antisense DNA inhibition of tumor growth induced by c-Ha-ras oncogene in nude mice". Cancer Res. 53:577, 1993), IFN-β (Du, W., and T. Maniatis: "An ATF/CREB binding site protein is required for virus induction of the human interferon B gene". Proc. Natl. Acad. Sci. USA 89:2150,1992), TGF-β1 (Asiedu, C.K., L. Scott, R.K. Assoian, M. Ehrlich: "Binding of AP-1/CREB proteins and of MDBP to contiguous sites downstream of the human TGF-B gene". Biochim. Biophys. Acta 1219:55,1994.), TGF-β2, class II MHC (Cox, P.M., and C.R. Goding: "An ATF/CREB binding motif is required for aberrant constitutive expression of the MHC class II DRa promoter and activation by SV40 T-antigen". Nucl. Acids Res. 20:4881,1992.), E-selectin, GM-CSF, CD-8α, the germline Igα constant region gene, the TCR Vβ gene, and the proliferating cell nuclear antigen (Huang, D., P.M. Shipman-Appasamy, D.J. Orten. S.H. Hinrichs, and M.B. Prystowsky: "Promoter activity of the proliferating-cell nuclear antigen gene is associated with inducible CRB-binding proteins in interleukin 2-stimulated T lymphocytes". Mol. Cell. Biol. 14:4233, 1994.). In addition to activation through the cAMP pathway, CREB can also mediate transcriptional responses to changes in intracellular Ca++ concentration (Sheng, M., G. McFadden, and M.E. Greenberg: "Membrane depolarization and calcium induce c-fos transcription via phosphorylation of transcription factor CREB". Neuron 4:571, 1990).
  • The role of protein-protein interactions in transcriptional activation by CREB/ATF proteins appears to be extremely important. Activation of CREB through the cyclic AMP pathway requires protein kinase A (PKA), which phosphorylates CREB341 on ser133 and allows it to bind to a recently cloned protein, CBP (Kwok, R.P.S., J.R. Lundblad, J.C. Chrivia, J.P. Richards, H.P. Bachinger, R.G. Brennan, S.G.E. Roberts, M.R. Green, and R.H. Goodman: "Nuclear protein CBP is a coactivator for the transcription factor CREB". Nature 370.223,1994; Arias, J., A.S. Alberts, P. Brindle, F.X Claret, T. Smea, M. Karin, J. Feramisco, and M. Montinmy: "Activation of cAMP and mitogen responsive genes relies on a common nuclear factor". Nature 370:226,1994.). CBP in turn interacts with the basal transcription factor TFIIB causing increased transcription. CREB also has been reportod to interact with dTAFII 110, a TATA binding protein-assodated factor whose binding may regulate transcription (Ferreri, K., G. Gill, and M. Montminy: "The cAMP-regulated transcription factor CREB interacts with a component of the TFIID complex". Proc. Natl. Acad. Sci. USA 91:1210, 1994.). In addition to these interactions, CREB/ATF proteins can specifically bind multiple other nuclear factors (Hoeffler, J.P., J.W. Lustbader, and C.-Y. Chen: "Identification of multiple nuclear factors that interact with cyclic adenosine 3',5'-monophosphate response element-binding protein and activating transcription factor-2 by protein-protein interactions". Mol. Endocrinol. 5:256, 1991) but the biologic significance of most of these interactions is unknown. CREB is normally thought to bind DNA either as a homodimer or as a heterodimer with several other proteins. Surprisingly, CREB monomers constitutively activate transcription (Krajewski, W., and K.A.W. Lee: "A monomeric derivative of the cellular transcription factor CREB functions as a constitutive activator". Mol. Cell. Biol. 14:7204,1994.).
  • Aside from their critical role in regulating cellular transcription, it has recently been shown that CREB/ATF proteins are subverted by some infectious viruses and retroviruses, which require them for viral replication. For example, the cytomegalovirus immediate early promoter, one of the strongest known mammalian promoters, contains eleven copies of the CRE which are essential for promoter function (Chang, Y.-N., S. Crawford, J. Stall, D.R. Rawlins, K.-T. Jeang, and G.S. Hayward: "The palindromic series I repeats in the simian cytomegalovirus major immediate-early promoter behave as both strong basal enhancers and cyclic AMP response elements". J. Virol. 64:264, 1990). At least some of the tmnscriptional activating effects of the adenovirus E1A protein, which induces many promoters, are due to its binding to the DNA binding domain of the CREB/ATF protein, ATF-2, which mediates E1A inducible transcription activation (Liu, F., and M.R. Green: "Promoter targeting by adenovirus Ela through interaction with different cellular DNA-binding domains". Nature 368-520,1994). It has also been suggested that E1A binds to the CREB-binding protein, CBP (Arany, Z., W.R. Sellers, D.M. Livingston, and R. Eckner "E1A-associated p300 and CREB-associated CBP belong to a conserved family of coactivators". Cell 77:799,1994). Human T lymphotropic virus-I (HTLV-1), the retrovirus which causes human T cell leukemia and tropical spastic paresis, also requires CREB/ATF proteins for replication. In this case, the retrovirus produces a protein. Tax, which binds to CREB/ATF proteins and redirects them from their normal cellular binding sites to different DNA sequences (flanked by G- and C-rich sequences) present within the HTLV transcriptional enhancer (Paca-Uccaralertkun, S., L.-J. Zhao, N. Adya, J.V. Cross. B.R. Cullen, I.M. Boros, and C.-Z. Giam: "In vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator. Tax". Mol. Cell. Biol. 14:456, 1994; Adya, N., L.-J. Zhao, W. Huang, I. Boros, and C.-Z. Giam: "Expansion of CREB's DNA recognition specificity by Tax results from interaction with Ala-Ala-Arg at positions 282-284 near the conserved DNA-binding domain of CREB". Proc. Natl. Acad Sci. USA 91:5642, 1994).
    • Summary of the Invention
  • The instant invention is based on the finding that certain oligonucleotides containing unmethylated cytosine-guanine (CpG) dinucleotides activate lymphoeytes as evidenced by in vitro and in vivo data. Based on this finding, the invention features, in one aspect, novel immunostimulatory oligonucleotide compositions.
  • Accordingly, in a first aspect, the present invention provides a composition, for use as a medicament, comprising a vaccine and a synthetic immunostimulatory oligonucleotide of at least 8 bases in size, which contains at least one unmethylated CpG dinucleotide and is an adjuvant for the vaccine.
  • In a second aspect the invention provides the use of a composition in accordance with the first aspect of the invention, for the manufacture of a medicament for vaccinating a subject.
  • In a further aspect, the invention provides the use of a synthetic immunostimulatory oligonucleotide of at least 8 bases in size, which contains at least one unmethylated CpG dinucleotide, for the manufacture of a medicament for administration to a subject in conjunction with a vaccine, as an adjuvant for the vaccine.
  • Preferred embodiments and aspects of the invention are described in the attached claims.
  • In a preferred embodiment, an immunostimulatory oligonucleotide is synthetic, between 8 to 100 bases in size and contains a consensus mitogenic CpG motif represented by the formula:

            5' X1X2CGX3X4 3'

    wherein C and G are unmethylated, X1, X2, X3 and X4 are nucleotides and a GCG trinuclootide sequence is not present at or near the 5' and 3' termini.
  • For facilitating uptake into cells, CpG containing immunostimulatory oligonucleotides are preferably in the range of 8 to 40 bases in size. Prolonged immunostimulation can be obtained using stabilized oligonucleotides, particularly phosphorothioate stabilized oligonucleotides. Enhanced immunostimulatory activity has been observed where X1X2 is the dinucleotide GpA and/or X3X4 is the dinucleotide is most preferably TpC or also TpT. Further enhanced immunostimulatory activity has been observed where the consensus motif X1X2CGX3X4 is preceded on the 5' end by a T.
  • In another aspect, the invention features useful methods, which are based on the immunostimulatory activity of the oligonucleotides. For example, lymphocytes can either be obtained from a subject and stimulated ex vivo upon contact with an appropriate oligonucleotide; or a non-methylated CpG containing oligonucleotide can be administered to a subject to facilitate in vivo activation of a subject's lymphocytes. Activated lymphocytes, stimulated by the methods described herein (e.g. either ex vivo or in vivo), can boost a subject's immune response. The immunostimulatory oligonucleotides can therefore be used to treat, prevent or ameliorate an immune system deficiency (e.g., a tumor or cancer or a viral, fungal, bacterial or parasitic infection in a subject In addition, immunostimulatory oligonucleotides can also be administered as a vaccine adjuvant, to stimulate a subject's response to a vaccine.
  • Other features and advantages of the invention will become more apparent from the following detailed description and claims.
    • Detailed Description of the Invention Definitions
  • As used herein, the following terms and phrases shall have the meanings set forth below:
  • An "oligonucleotide" or "oligo" shall mean multiple nucleotides (i.e. molecules comprising a sugar (e.g. ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g. cytosine (C), thymine (T) or uracil (U)) or a substituted purine (e.g. adenine (A) or guanine (G)). The term "oligonucleotide'' as used herein refers to both oligoribonucleotides (ORNs) and oligodeoxyribonucleotides (ODNs). The term "oligonucleotide" shall also include oligonucleosides (i.e. an oligonucleotide minus the phosphate) and any other organic base containing polymer. Oligonucleotides can be obtained from existing nucleic acid sources (e.g. genomic or cDNA), but are preferably synthetic (e.g. produced by oligonucleotide synthesis).
  • A "stabilized oligonucleotide" shall mean an oligonucleotide that is relatively resistant to in vivo degradation (e.g. via an exo- or endo-nuclease). Preferred stabilized oligonucleotides of the instant invention have a modified phosphate backbone. Especially preferred oligonucleotides have a phosphorothioate modified phosphate backbone (i.e. at least one of the phosphate oxygens is replaced by sulfur). Other stabilized oligonucleotides include: nonionic DNA analogs, such as alkyl- and aryl- phosphonates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), phosphodiester and alkylphosphotriesters, in which the charged oxygen moiety is alkylated. Oligonucleotides which contain a diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini have also been shown to be substantially resistant to nuclease degradation.
  • An "immunostimulatory oligonucleotide", "immunostimulatory CpG containing oligonucleotide", or "CpG ODN" refer to an oligonucleotide, which contains a cytosine, guanine dinucleotide sequence and stimulates (e.g. has a mitogenic effect) on vertebrate lymphocytes. Preferred immunostimulatory oligonucleotides are between 8 to 100 bases in size and contain a consensus mitogenic CpG motif represented by the formula:

            5' X1X2CGX3X4 3'

    wherein C and G are unmethylated, X1, X2, X3 and X4 are nucleotides and a GCG trinucleotide sequence is not present at or near the 5' and 3' termini.
  • Preferably the immunostimulatory oligonucleotides range between 8 to 40 bases in size. In addition, the immunostimulatory oligonucleotides are preferably stabilized oligonucleotides, particularly preferred are phosphorothioate stabilized oligonucleotides. In one preferred embodiment, X1X2 is the dinucleotide GpA. In another preferred embodiment, X3X4 is preferably the dinucleotide TpC or also TpT. In a particularly preferred embodiment, the consensus motif X1X2CGX3X4 is preceded on the 5' end by a T. Particularly preferred consensus sequences are TGACGTT or TGACGTC.
  • Prolonged immunostimulation can be obtained using stabilized oligonucleotides, particularly phosphorothioate stabilized.
  • "Palindromic sequence" shall mean an inverted repeat (i.e. a sequence such as ABCDEE'D'CB'A' in which A and A' are bases capable of forming the usual Watson-Crick base pairs. In vivo, such sequences may form double stranded structures.
  • An "oligonucleotide delivery complex" shall mean an oligonucleotide associated with (e.g. ionically or covalently bound to; or encapsulated within) a targeting means (e.g. a molecule that results in higher affinity binding to target cell (e.g. B-cell and natural killer (NK) cell) surfaces and/or increased cellular uptake by target cells). Examples of oligonucleotide delivery complexes include oligonucleotides associated with: a sterol (e.g. cholesterol), a lipid (e.g. a cationic lipid, virosome or liposome), or a target cell specific binding agent (e.g. a ligand recognized by target cell specific receptor). Preferred complexes must be sufficiently stable in vivo to prevent significant uncoupling prior to internalization by the target cell. However, the complex should be cleavable under appropriate conditions within the cell so that the oligonucleotide is released in a functional form.
  • An "immune system deficiency" shall mean a disease or disorder in which the subject's immune system is not functioning in normal capacity or in which it would be useful to boost a subject's immune response for example to eliminate a tumor or cancer (e.g. tumors of the brain, lung (e.g. small cell and non-small cell), ovary, breast, prostate, colon, as well as other carcinomas and sarcomas) or a viral (e.g. HIV, herpes), fungal (e.g. Candida sp.), bacterial or parasitic (e.g. Leishmania, Toxoplasma) infection in a subject.
  • A "disease associated with immune system activation" shall mean a disease or condition caused or exacerbated by activation of the subject's immune system. Examples include systemic lupus erythematosus, sepsis and autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.
  • A "subject" shall mean a human or vertebrate animal including a dog, cat, horse, cow, pig, sheep, goat, chicken, monkey, rat, mouse, etc.
    • Certain Unmethylated CpG Containing Oligos Have B Cell Stimulatory Activity As Shown in vitro and in vivo
  • In the course of investigating the lymphocyte stimulatory effects of two antisense oligonucleotides specific for endogenous retroviral sequences, using protocols described in the attached Examples 1 and 2, it was surprisingly found that two out of twenty-four "controls" (including various scrambled, sense, and mismatch controls for a panel of "antisense" ODN) also mediated B cell activation and IgM secretion, while the other "controls" had no effect.
  • Two observations suggested that the mechanism of this B cell activation by the "control" ODN may not involve antisense effects 1) comparison of vertebrate DNA sequences listed in GenBank showed no greater homology than that seen with non-stimulatory ODN and 2) the two controls showed no hybridization to Northern blots with 10 µg of spleen poly A+ RNA. Resynthesis of these ODN on a different synthesizer or extensive purification by polyacrylamide gel electrophoresis or high pressure liquid chromatography gave identical stimulation, eliminating the possibility of an impurity. Similar stimulation was seen using B cells from C3H/HeJ mice, eliminating the possibility that lipopolysaccharide (LPS) contamination could account for the results.
  • The fact that two "control" ODN caused B cell activation similar to that of the two "antisense" ODN raised the possibility that all four ODN were stimulating B cells through some non-antisense mechanism involving a sequence motif that was absent in all of the other nonstimulatory control ODN. In comparing these sequences, it was discovered that all of the four stimulatory ODN contained ODN dinucleotides that were in a different sequence context from the nonstimulatory control.
  • To determine whether the CpG motif present in the stimulatory ODN was responsible for the observed stimulation, over 300 ODN ranging in length from 5 to 42 bases that contained methylated, unmethylated, or no CpG dinucleotides in various sequence contexts were synthesized. These ODNs, including the two original "controls" (ODN 1 and 2) and two originally synthesized as "antisense" (ODN 3D and 3M; Krieg, A.M. J. Immunol. 143:2448 (1989)), were then examined for in vitro effects on spleen cells (representative sequences are listed in Table 1). Several ODN that contained CpG dinucleotides induced B cell activation and IgM secretion; the magnitude of this stimulation typically could be increased by adding more CpG dinucleotides (Table 1; compare ODN 2 to 2a or 3D to 3Da and 3Db). Stimulation did not appear to result from an antisense mechanism or impurity. ODN caused no detectable activation of γδ or other T cell populations.
  • Mitogenic ODN sequences uniformly became nonstimulatory if the CpG dinucleotide was mutated (Table 1; compare ODN 1 to 1a; 3D to 3Dc; 3M to 3Ma; and 4 to 4a) or if the cytosine of the CpG dinucleotide was replaced by 5-methylcytosine (Table 1; ODN 1b,2b,2c,3Dd, and 3Mb). In contrast, methylation of other cytosines did not reduce ODN activity (ODN 1c,2d. 3De and 3Mc). These data confirmed that a CpG motif is the essential element present in ODN that activate B cells.
  • In the course of these studies, it became clear that the bases flanking the CpG dinucleotide played an important role in determining the B cell activation induced by an ODN. The optimal stimulatory motif was determined to consist of a CpG flanked by two 5' purines (preferably a GpA dinucleotide) and two 3' pyrimidines (preferably a TpT or TpC dinucleotide). Mutations of ODN to bring the CpG motif closer to this ideal improved stimulation (e.g. compare ODN 2 to 2e; 3M to 3Md) while mutations that disturbed the motif reduced stimulation (e.g. compare ODN 3D to 3Df; 4 to 4b, 4c and 4d). On the other hand, mutations outside the CpG motif did not reduce stimulation (e.g. compare ODN 1 to 1d; 3D to 3Dg; 3M to 3Me).
  • Of those tested, ODNs shorter than 8 bases were non-stimulatory (e.g. ODN 4e). Among the forty-eight 8 base ODN tested. the most stimulatory sequence identified was TCAACGTT (ODN 4) which contains the self complementary "palindrome" AACGTT. In further optimizing this motif, it was found that ODN containing Gs at both ends showed increased stimulation, particularly if the the ODN were rendered nuclease resistant by phosphorothioate modification of the terminal intemucleotide linkages. ODN 1585 (5' GGGGTCAACGTTCAGGGGGG 3' (SEQ ID NO:1)), in which the first two and last five internucleotide linkages are phosphorothioate modified caused an average 25.4 fold increase in mouse spleen cell proliferation compared to an average 3.2 fold increase in proliferation induced by ODN 1638, which has the same sequence as ODN 1585 except that the 10 Gs at the two ends are replaced by 10 As. The effect of the G-rich ends is cis; addition of an ODN with poly G ends but no CpG motif to cells along with 1638 gave no increased proliferation.
  • Other octamer ODN containing a 6 base palindrome with a TpC dinucleotide at the 5' end were also active if they were dose to the optimal motif (e.g. ODN 4b,4c). Other dinucleotides at the 5' end gave reduced stimulation (eg ODN 4f; all sixteen possible dinucleotides were tested). The presence of a 3' dinucleotide was insufficient to compensate for the lack of a 5' dinucleotide (eg. ODN 4g). Disruption of the palindrome eliminated stimulation in octamer ODN (eg., ODN 4h), but palindromes were not required in longer ODN. Table 1: Oligonucleotide Stimulation of B Cells
    Stimulation Index'
    ODN Sequence (5' to 3')† 3H Uridine IgM Production
    1 (SEQ ID NO: 2) GCTAGACGTTAGCGT: 6.1 ± 0.8 17.9 ± 3.6
    1a (SEQ. ID NO: 3) ......T........ 1.2 ± 0.2 1.7 ± 0.5
    1b (SEQ ID NO: 4) ......Z........ 1.2 ± 0.1 1.8 ± 0.0
    1c (SEQ ID NO: 5) ............Z.. 10.3 ± 4.4 9.5 ± 1.8
    1d (SEQ ID NO: 6) ..AT......GAGC. 13.0 ± 2.3 18.3 ± 7.5
    2 (SEQ ID NO: 7) ATGGAAGGTCCAGCGTTCTC 2.9 ± 0.2 13.6 ± 2.0
    2a (SEQ ID NO: 8) ..C..CTC..G......... 7.7 ± 0.8 24.2 ± 3.2
    2b (SEQ ID NO: 9) ..Z..CTC.ZG..Z...... 1.6 ± 0.5 2.8 ± 2.2
    2e(SEQ ID NO: 10) ..Z..CTC..G............ 3.1±0.6 7.3 ± 1.4
    2d (SEQ ID NO: 11) ..C...CTC..G......Z.. 7.4±1.4 27.7±5.4
    2e (SEQ ID NO: 12) ............A........ 5.6±2.0 ND
    3D (SEQ ID NO: 13) GAGAACGCTGGACCTTCCAT 4.9±0.5 19.9±3.6
    3Da (SEQ ID NO:14) ...........C........... 66±1.5 33.9±6.9
    3Db (SEQ ID NO: I5) .............C........G... 10.1±2.8 25.4±0.8
    3Dc (SEQ ID NO: 16) ... C.A.............. 1.0±0.1 1.2±0.5
    3Dd (SEQ ID NO: 17) .....z.............. 12±02 1.0±0.4
    3De (SEQ ID NO: 18) ...............z...... 4.4±12 18.8±4.4
    3Df (SEQ ID NO: 19) .......A............ 1.6±0.1 7.7±0.4
    3Dg (SEQ ID NO: 20) ..........CC.G.ACTG.. 6.1±1.5 18.6±1.5
    3M (SEQ ID NO: 21) TCCATGTCGGTCCTGATGCT 4.1 ± 02 232±4.9
    3Ma (SEQ ID NO:22) .....CT............ 0.9±0.1 1.8±0.5
    3Mb(SEQ ID NO:23) .....Z.............. 1.3± 0.3 1.5±0.6
    3Mc (SEQ ID NO:24) ............Z........ 5.4±1.5 8.5±2.6
    3Md (SEQ ID NO: 25) .....A..T............ 17.2±9.4 ND
    3Me (SEQ ID NO: 26) ...............C..A. 3.6±02 142±5.2
    4 TCAACGTT 6.1 ± 1.4 19.2 ± 5.2
    4a ....GC.. 1. 1 ± 0.2 15 ± 1.1
    4b ...GCGC. 4.5.± 0.2 9.6 ± 3.4
    4c ...TCGA. 2.7.± 1.0 ND
    4d .. TT..AA 1.3 ± 0.2 ND
    4e -....... 1.3 ± 0.2 1.1 ± 0.5
    4f c....... 3.9 ± 1.4 ND
    4g --......CT 1.4 ± 0.3 ND
    4h .......C 1.2 ± 02 ND
    LPS 7.8 ± 2.5 4.8 ± 1.0
    ' Stimulation indexes are the means and std. dev. derived from at least 3 separate experiments, and are compared to wells cultured with no added ODN. ND - not done. CpG dinucleotides are underlined. Dots indicate identity; dashes indicate deletions. Z indicates 5 methyl cytosine.)
  • The kinetics of lymphocyte activation were investigated using mouse spleen cells. When the cells were pulsed at the same time as ODN addition and harvested just four hours later, there was already a two-fold increase in 3H uridine incorporation. Stimulation peaked at 12-48 hours and then decreased. After 24 hours, no intact ODN were detected, perhaps accounting for the subsequent fall in stimulation when purified B cells with or without anti-IgM (at a submitogenic dose) were cultured with CpG ODN, proliferationwas found to synergistically increase about 10-fold by the two mitogens in combination after 48 hours. The magnitude of stimulation was concentration dependent and consistently exceeded that of LPS under optimal conditions for both. Oligonucleotides containing a nuclease resistant phosphorothioate backbone were approximately two hundred times more potent than unmodified oligonucleotides.
  • Cell cycle analysis was used to determine the proportion of B cells activated by CpG-ODN. CpG-ODN induced cycling in more than 95% of B cells (Table 2). Splenic B lymphocytes sorted by flow cytometry into CD23- (marginal zone) and CD23+ (follicular) subpopulations were equally responsive to ODN- induced stimulation, as were both resting and activated populations ofB cells isolated by fractionation over Percoll gradients. These studies demonstrated that CpG-ODN induce essentially all B cells to enter the cell cycle. Table 2: Cell Cycle Analysis with CpG ODN
    Percent of cells in
    Treatment G0 G1 SA+G2+M
    Media 97.6 2.4 0.02
    ODN 1a a 95.2 4.8 0.04
    ODN 1d 2.7 74.4 22.9
    ODN 3Db 3.5 76.4 20.1
    LPS (30 µg/ml) 17.3 70.5 12.2
  • The mitogenic effects of CpG ODN on human cells, were tested on peripheral blood mononuclear cells (PBMCs) obtained from two patients with chronic lymphocytic leukemia (CLL), as described in Example 1. Control ODN containing no CpG dinucleotide sequence showed no effect on the basal proliferation of 442 cpm and 874 cpm (proliferation measured by 3H thymidine incorporation) of the human cells. However, a phosphorothioate modified CpG ODN 3Md (SEQ m NO: 25) induced increased proliferation of 7210 and 86795 cpm respectively in the two patients at a concentration of just 1 µM. Since these cells had been frozen, they may have been less responsive to the oligos than fresh cells in vivo In addition, cells from CLL patients typically are non-proliferating, which is why traditional chemotherapy is not effective.
  • Certain B cell lines such as WEHI-231 are induced to undergo growth arrest and/or apoptosis in response to crosslinking of their antigen receptor by anti-IgM (Jakway, J.P. et al., "Growth regulation of the B lymphoma cell line WEHI-231 by anti-immunoglobulin, lipopolysaccharide and other bacterial products" J. Imminol. 137: 2225 (1986); Tsubata, T., J. Wu and T. Honjo: B-cell apoptosis induced by antigen receptor crosslinking is blocked by a T-cell signal through CD40." Nature 364: 645 (1993)). WEHI-231 cells are rescued from this growth arrest by certain stimuli such as LPS and by the CD40 ligand. ODN containing the CpG motif were also found to protect WEHI-231 from anti-IgM induced growth arrest, indicating that accessory cell populations are not required for the effect.
  • To better understand the immune effects of unmethylated CpG ODN, the levels of cytokines and prostaglandins in vitro and in vivo were measured. Unlike LPS, CpG ODN were not found to induce purified macrophages to produce prostaglandin PGE2. In fact, no apparent direct effect of CpG ODN was detected on either macrophages or T cells. In vivo or in whole spleen cells, no significant increase in the following interleukins: IL-2. IL-3, IL-4, or IL-10 was detected within the first six hours. However, the level of IL-6 increased strikingly within 2 hours in the serum of mice injected with CpG ODN. Increased expression of IL-12 and interferon gamma (EFN-γ) by spleen cells was also detected within the first two hours.
  • To determine whether CpG ODN can cause in vivo immune stimulation, DBA/2 mice were injected once intraperitoneally with PBS or phosphorothioate CpG or non-CpG ODN at a dose of 33 mg/kg (approximately 500 µg/mouse). Pharmacokinetic studies in mice indicate that this dose of phosphorothioate gives levels of approximately 10 µg/g in spleen tissue (within the effective concentration range determined from the in vitro studies described herein) for longer than twenty-four hours (Agrawal, S. et al. (1991) Proc. Natl. Acad Sci. USA 91:7595). Spleen cells from mice were examined twenty-four hours after ODN injection for expression ofB cells surface activation markers Ly-6A/E, Bla-1, and class II MHC using three color flow cytometry and for their spontaneous proliferation using 3H thymidine. Expression of all three activation markers was significantly increased in B cells from mice injected with CpG ODN, but not from mice injected with PBS or non-CpG ODN. Spontaneous 3H thymidine incorporation was increased by 2-6 fold in spleen cells from mice injected with the stimulatory ODN compared to PBS or non-CpG ODN-injected mice. After 4 days, serum IgM levels in mice injected with CpG ODN in vivo were increased by approximately 3-fold compared to controls. Consistent with the inability of these agents to activate T cells, there was minimal change in T cell expression of the IL-2R or CD-44.
  • Degradation of phophodiester ODN in serum is predommantly mediated by 3' exonucleases, while intracellular ODN degradation is more complex, involving 5' and 3' exonucleases and endonucleases. Using a panel of ODN bearing the 3D sequence with varying numbers of phosphorothioate modified linkages at the 5' and 3' ends, it was empirically determined that two 5' and five 3' modified linkages are required to provide optimal stimulation with this CpG ODN.
    • Unmethylated CpG Containing Oligos Have NK Cell Stimulatory Activity
  • As described in further detail in Example 4, experiments were conducted to determine whether CpG containing oligonucleotides stimulated the activity of natural killer (NK) cells in addition to B cells. As shown in Table 3, a marked induction ofNK activity among spleen cells cultured with CpG ODN 1 and 3Dd was observed. In contrast, there was relatively no induction in effectors that had been treated with non-CpG control ODN. Table 3: Induction Of NK Activity By CpG Oligodeoxynucleotides (ODN)
    % YAC-1 Specific Lysis* % 2C11 Specific Lysis
    Effector: Target Effector: Target
    ODN 50:1 100:1 50:1 100:1
    None -1.1 -1.4 15.3 16.6
    1 16.1 24.5 38.7 47.2
    3Dd 17.1 27.0 37.0 40.0
    non-CpG ODN -1.6 -1.7 14.8 15.4
    • Neutralizing Activity of Methylated CpG Containing Oligos
  • B cell mitogenicity of ODN in which cytosines in CpG motifs or elsewhere were replaced by 5-methylcytosine were tested as described in Example 1. As shown in Table 1 above, ODN containing methylated CpG motifs were non-mitogenic (Table 1; ODN 1c, 2f, 3De, and 3Mc). However, methylation of cytosines other than in a CpG dinucleotide retained their stimulatory properties (Table 1, ODN 1d, 2d, 3Df, and 3Md).
    • Immoinhibitory Activity of Oligos Containing a GCG Trinucleotide Sequence at or near both termini
  • In some cases, ODN containing CpG dinucleotides that are not in the stimulatory motif described above were found to block the stimulatory effect of other mitogenic CpG ODN. Specifically the addition of an atypical CpG motif consisting of a GCG near or at the 5' and/or 3' end of CpG ODN actually inhibited stimulation of proliferation by other CpG motifs. Methylation or substitution of the cytosine in a GCG motif reverses this effect. By itself, a GCG motif in an ODN has a modest mitogenic effect, though far lower than that seen with the preferred CpG motif.
    • Proposed Mechanisms of Action of Immunostimulatory, Neutralizing and Immunoinhibitory Oligonucleotides
  • Unlike antigens that trigger B cells through their surface Ig receptor, CpG-ODN did not induce any detectable Ca2+ flux, changes in protein tyrosine phosphorylation, or IP 3 generation. Flow cytometry with FITC-conjugated ODN with or without a CpG motif was performed as described in Zhao, Q et al.,(Antisense Research and Development 3:53-66 (1993)), and showed equivalent membrane binding, cellular uptake, efflux, and intracellular localization. This suggests that there may not be cell membrane proteins specific for CpG ODN. Rather than acting through the cell membrane, that data suggests that unmethylated CpG containing oligonucleotides require cell uptake for activity: ODN covalently linked to a solid Teflon support were nonstimulatory, as were biotinylated ODN immobilized on either avidin beads or avidin coated petri dishes. CpG ODN conjugated to either FITC or biotin retained full mitogenic properties, indicating no steric hindrance.
  • The optimal CpG motif (TGACGTT/C is identical to the CRE (cyclic AMP response element). Like the mitogenic effects of CpG ODN, binding of CREB to the CRE is abolished if the central CpG is methylated. Electrophoretic mobility shift assays were used to determine whether CpG ODN, which are single stranded, could compete with the binding of B cell CREB/ATF proteins to their normal binding site, the doublestranded CRE. Competition assays demonstrated that single stranded ODN containing CpG motifs could completely compete the binding of CREB to its binding site, while ODN without CpG motifs could not. These data support the conclusion that CpG ODN exert their mitogenic effects through interacting with one or more B cell CREB/ATF proteins in some way. Conversely, the presence of GCG sequences or other atypical CPG motifs near the 5' and/or 3' ends of ODN likely interact with CREB/ATF proteins in a way that does not cause activation, and may even prevent it.
  • The stimulatory CpG motif is common in microbial genomic DNA, but quite rare in vertebrate DNA. In addition, bacterial DNA has been reported to induce B cell proliferation and immunoglobulin (Ig) production, while mammalian DNA does not (Messina, J.P. et al., J Immunol. 147:1759 (1991)). Experiments further described in Example 3, in which methymon of bacterial DNA with CpG methylase was found to abolish mitogenicity, demonstrates that the difference in CpG status is the cause of B cell stimulation by bacterial DNA. This data supports the following conclusion: that umnethylated CpG dinucleotides present within bacterial DNA are responsible for the stimulatory effects of bacterial DNA.
  • Teleologically, it appears likely that lymphocyte activation by the CpG motif represents an immune defense mechanism that can thereby distinguish-bacterial from host DNA. Host DNA would induce little or no lymphocyte activation due to it CpG suppression and methylation. Bacterial DNA would cause selective lymphocyte activation in infected tissues. Since the CpG pathway synergizes with B cell activation through the antigen receptor, B cells bearing antigen receptor specific for bacterial antigens would receive one activation signal through cell membrane Ig and a second signal from bacterial DNA, and would therefore tend to be preferentially activated. The interrelationship of this pathway with other pathways of B cell activation provide a physiologic mechanism employing a polyclonal antigen to induce antigen-specific responses.
    • Method for Making Immunostimulatory Oligos
  • For use in the instant invention, oligonucleotides can be synthesized de novo using any of a number of procedures well known in the art. For example, the β-cyanoethyl phosphoramidite method (S.L. Beaucage and M.H. Caruthers, (1981) Tet. Let. 22:1859); nucleoside H-phosphonate method (Garegg et al., (1986) Tet. Let. 27: 4051-4054; Froehler et al., (1986) Nucl. Acid Res. 14: 5399-5407; Garegg et al., (1986) Tet. Let. 27: 4055-4058, Gaffney et al., (1988) Tet. Let. 29:2619-2622). These chemistries can be performed by a variety of automated oligonucleotide synthesizers available in the market. Alternatively, oligonucleotides can be prepared from existing nucleic acid sequences (e.g. genomic or cDNA) using known techniques, such as those employing restriction enzymes, exonucleases or endonucleases.
  • For use in vivo, oligonucleotides are preferably relatively resistant to degradation (e.g. via endo- and exo- nucleases). Oligonucleotide stabilization can be accomplished via phosphate backbone modifications. A preferred stabilized oligonucleotide has a phosphorothioate modified backbone. The pharmacokinetics of phosphorothioate ODN show that they have a systemic half-life of forty-eight hours in rodents and suggest that they may be useful for in vivo applications (Agrawal, S. et al. (1991) Proc. Natl. Acad Sci. USA 88:7595). Phosphorothioates may be synthesized using automated techniques employing either phosphoramidate or H phosphonate chemistries. Aryl- and alkyl- phosphonates can be made e.g. (as described in U.S. Patent No. 4,469,863 ); and alkylphosphotriesters (in which the charged oxygen moiety is alkylated as described in U.S. Patent No. 5,023,243 and European Patent No. 092,574 ) can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other DNA backbone modifications and substitutions have been described (Uhlmann, E. and Peyman, A. (1990) Chem Rev. 90:544; Goodchild, J. (1990) Bioconjugate Chem. 1:165).
  • For administration in vivo, oligonucleotides may be associated with a molecule that results in higher affinity binding to target cell (e.g. B-cell and natural killer (NK) cell) surfaces and/or increased cellular uptake by target cells to form an "oligonucleotide delivery complex". Oligonucleotides can be ionically, or covalently associated with appropriate molecules using techniques which are well known in the art. A variety of coupling or crosslinking agents can be used e.g. protein A, carbodiimide, and N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). Oligonucleotides can alternatively be encapsulated in liposomes or virosomes using well-known techniques.
  • The present invention is further illustrated by the following Examples which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.
    • Therapeutic Uses of Immunostimulatory Oligos
  • Based on their immunostimulatory properties, oligonucleotides containing at least one unmethylated CpG dinucleotide can be administered to a subject in vivo to treat an "immune system deficiency". Alternatively, oligonucleotides containing at least one unmethylated CpG dinucleotide can be contacted with lymphocytes (e.g. B cells or NK cells) obtained from a subject having an immune system deficiency ex vivo and activated lymphocytes can then be reimplanted in the subject.
  • Immunostimulatory oligonucleotides can be administered to a subject in conjunction with the vaccine, as an adjuvant, to boost a subject's immune system to effect better response from the vaccine, slightly before or at the same time as the vaccine.
  • CpG ODN also increased natural killer cell activity in both human and murine cells. Induction of NK activity may likewise be beneficial in cancer immunotherapy.
  • For use in therapy, an effective amount of an appropriate oligonucleotide alone or formulated as an oligonucleotide delivery complex can be administered to a subject by any mode allowing the oligonucleotide to be taken up by the appropriate target cells (e.g. B-cells and NK cells). Preferred routes of administration include oral and transdermal (e.g. via a patch). Examples of other routes of administration include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, etc.). The injection can be in a bolus or a continuous infusion.
  • An oligonucleotide alone or as an oligonucleotide delivery complex can be administered in conjunction with a pharmaceutically acceptable carrier. As used herein, the phrase "pharmaceutically acceptable carrier" is intended to include substances that can be coadministered with an oligonucleotide or an oligonucleotide delivery complex and allows the oligonucleotide to perform its intended function. Examples of such carriers include solutions, solvents, dispersion media, delay agents, emulsions and the like. The use of such media for pharmaceutically active substances are well known in the art. Any other conventional carrier suitable for use with the oligonucleotides falls within the scope of the instant invention.
  • The language "effective amount" of an oligonucleotide refers to that amount necessary or sufficient to realize a desired biologic effect. For example, an effective amount of an oligonucleotide containing at least one unmethylated CpG for treating an immune system deficiency could be that amount necessary to eliminate a tumor, cancer, or bacterial, viral or fungal infection. An effective amount for use as a vaccine adjuvant could be that amount useful for boosting a subject's immune response to a vaccine. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular oligonucleotide being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular oligonucleotide without necessitating undue experimentation.
  • The studies reported above indicate that unmethylated CpG containing oligonucleotides are directly mitogenic for lymphocytes (e.g. B cells and NK cells). Together with the presence of these sequences in bacterial DNA, these results suggest that the underrepresentation of CpG dinucleotides in animal genomes, and the extensive methylation of cytosines present in such may be explained by the existence of an immune defense mechanism that can distinguish bacterial from host DNA. Host DNA would commonly be present in many anatomic regions and areas of inflammation due to apoptosis (cell death), but generally induces little or no lymphocyte activation. However, the presence of bacterial DNA containing umnethylated CpG motifs can cause lymphocyte activation precisely in infected anatomic regions, where it is beneficial. This novel activation pathway provides a rapid alternative to T cell dependent antigen specific B cell activation. However, it is likely that B cell activation would not be totally nonspecific. B cells bearing antigen receptors specific for bacterial products could receive one activation signal through cell membrane Ig, and a second from bacterial DNA, thereby more vigorously triggering antigen specific immune responses.
    • Therapeutic Uses of Oligos Containing GCG Trinucleotide Sequences at or Near Both Termini
  • Based on their interaction with CREB/ATF, oligonucleotides containing GCG trinucleotide sequences at or near both termini have antiviral activity, independent of any antisense effect due to complementarity between the oligonucleotide and the viral sequence being targeted. Based on this activity, an effective amount of inhibitory oligonucleotides can be administered to a subject to treat or prevent a viral infection.
    • EXAMPLES Example 1: Effects of ODNs on B Cell Total RNA Synthesis and Cell Cycle
  • B cells were purified from spleens obtained from 6-12 wk old specific pathogen free DBA/2 or BXSB mice (bred in the University of Iowa animal care facility; no substantial strain differences were noted) that were depleted ofT cells with anti-Thy-1.2 and complement and centrifugation over lympholyte M (Cedarlane Laboratories, Hornby, Ontario, Canada) ("B cells"). B cells contained fewer than 1% CD4+ or CD8+ cells. 8x104 B cells were dispensed in triplicate into 96 well microtiter plates in 100 µl RPMI containing 10% FBS (heat inactivated to 65°C for 30 min.), 50 µM 2-mercaptoethanol, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamate. 20 µM ODN were added at the start of culture for 20 h at 37°C, cells pulsed with 1 µCi of 3H uridine, and harvested and counted 4 hr later. Ig secreting B cells were enumerated using the ELISA spot assay after culture of whole spleen cells with ODN at 20 µM for 48 hr. Data, reported in Table 1, represent the stimulation index compared to cells cultured without ODN. Cells cultured without ODN gave 687 cpm, while cells cultured with 20 µg/ml LPS (determined by titration to be the optimal concentration) gave 99,699 cpm in this experiment. 3H thymidine incorporation assays showed similar results, but with some nonspecific inhibition by thymidine released from degraded ODN (Matson. S and A.M. Krieg (1992) Nonspecific suppression of 3H-thymidine incorporation by control oligonucleotides. Antisense Research and Development 2:325).
  • For cell cycle analysis, 2 X 106 B cells were cultured for 48 hr. in 2 ml tissue culture medium alone, or with 30 µg/ml LPS or with the indicated phosphorothioate modified ODN at 1 µM. Cell cycle analysis was performed as described in (Darzynkiewicz, Z. et al., Proc. Natl. Acad Sci. USA 78 :2881 (1981)).
  • To test the mitogenic effects of CpG ODN on human cells, perpheral blood monocyte cells (PBMCs) were obtained from two patients with chronic lymphocytic leukemia (CLL), a disease in which the circulating cells are malignant B cells. Cells were cultured for 48 hrs and pulsed for 4 hours with tritiated thymidine as described above.
    • Example 2: Effects of ODN on Production of IgM from B cells
  • Single cell suspensions from the spleens of freshly killed mice were treated with anti-Thyl, anti-CD4, and anti-CD8 and complement by the method of Leibson et al., J. Exp. Med 154:1681 (1981)). Resting B cells (<,02% T cell contamination) were isolated from the 63 - 70% band of a discontinuous Percoll gradient by the procedure of DeFranco et al, J. Exp. Med 155:1523 (1982). These were cultured as described above in 30 µM ODN or 20 µg/ml LPS for 48 hr. The number ofB cells actively secreting IgM was maximal at this time point, as determined by ELIspot assay (Klinman, D.M. et al. J. Immunol. 144:506 (1990)). In that assay, B cells were incubated for 6 hrs on anti-Ig coated microtiter plates. The Ig they produced (>99% IgM) was detected using phosphatase-labelled anti-Ig (Southern Biotechnology Associated, Birmingham, AL). The antibodies produced by individual B cells were visualized by addition of BCIP (Sigma Chemical Co., St. Louis MO) which forms an insoluble blue precipitate in the presence of phosphatese. The dilution of cells producing 20 - 40 spots/well was used to determine the total number of antibody-secreting B cells/sample. All assays were performed in triplicate. In some experiments, culture supernatants were assayed for IgM by ELISA, and showed similar increases in response to CpG-ODN.
    • table 1 Example 3: B cell Stimulation by Bacterial DNA
  • DBA/2 B cells were cultured with no DNA or 50 µg/ml of a) Micrococcus lysodeikticus; b) NZB/N mouse spleen; and c) NFS/N mouse spleen genomic DNAs for 48 hours, then pulsed with 3H thymidine for 4 hours prior to cell harvest. Duplicate DNA samples were digested with DNAse I for 30 minutes at 37 C prior to addition to cell cultures. E coli DNA also induced an 8.8 fold increase in the number of IgM secreting B cells by 48 hours using the ELISA-spot assay.
  • DBA/2 B cells were cultured with either no additive, 50 µg/ml LPS or the ODN 1; 1a; 4; or 4a at 20 uM. Cells were cultured and harvested at 4, 8, 24 and 48 hours. BXSB cells were cultured as in Example 1 with 5, 10, 20,40 or 80 µM of ODN 1; 1a; 4; or 4a or LPS. In this experiment, wells with no ODN had 3833 cpm. Each experiment was performed at least three times with similar results. Standard deviations of the triplicate wells were <5%.
    • Example 4: Effects of ODN on natural killer (NK) activity
  • 10 x 106 C57BL/6 spleen cells were cultured in two ml RPMI (supplemented as described for Example 1) with or without 40 µM CpG or non-CpG ODN for forty-eight hours. Cells were washed, and then used as effector cells in a short term 51Cr release assay with YAC-1 and 2C11, two NK sensitive target cell lines (Ballas, Z. K. et al. (1993) J. Immunol. 150:17). Effector cells were added at various concentrations to 104 51Cr-labeled target cells in V-bottom microtiter plates in 0.2 ml, and incubated in 5% CO2 for 4 hr. at 37°C. Plates were then centrifuged, and an aliquot of the supernatant counted for radioactivity. Percent specific lysis was determined by calculating the ratio of the 51Cr released in the presence of effector cells minus the 51Cr released when the target cells are cultured alone, over the total counts released after cell lysis in 2% acetic acid minus the 51Cr cpm released when the cells are cultured alone.
    • Example 5: In vivo studies with CpG phosphorothioate ODN
  • Mice were weighed and injected IP with 0.25 ml of sterile PBS or the indicated phophomthioate ODN dissolved in PBS. Twenty four hours later, spleen cells were harvested, washed, and stained for flow cytometry using phycoerythrin conjugated 6B2 to gate on B cells in conjunction with biotin conjugated anti Ly-6A/E or anti-Iad (Pharmingen, San Diego, CA) or anti-Bla-1 (Hardy, R.R. et al., J. Exp. Med. 159:1169 (1984). Two mice were studied for each condition and analyzed individually.
    • Example 6 Titration of Phosphorothioate ODN for B Cell Stimulation
  • B cells were cultured with phosphorothioate ODN with the sequence of control ODN 1a or the CpG ODN 1d and 3Db and then either pulsed after 20 hr with 3H uridine or after 44 hr with 3H thymidine before harvesting and determining cpm.
    • Example 7 Rescue of B Cells From Apoptosis
  • WEHI-231 cells (5 x 104/well) were cultured for 1 hr. at 37 C in the presence or absence ofLPS or the control ODN 1a or the CpG ODN 1d and 3Db before addition of anti-IgM (1µ/ml). Cells were cultured for a further 20 hr. before a 4 hr. pulse with 2 µCi/well 3H thymidine. In this experiment, cells with no ODN or anti-IgM gave 90.4 x 103 by addition of anti-IgM. The phosphodiester ODN shown in Table 1 gave similar protection, though with some nonspecific suppression due to ODN degradation. Each experiment was repeated at least 3 times with similar results.
    • Example 8 In vivo induction of IL-6
  • DBA/2 female mice (2 mos. old) were injected IP with 500µg CpG or control phosphorothioate ODN. At various time points after injection, the mice were bled. Two mice were studied for each time point. IL-6 was measured by Elisa, and IL-6 concentration was calculated by comparison to a standard curve generated using recombinant EL-6. The sensitivity of the assay was 10 pg/ml. Levels were undetectable after 8 hr.
    • Example 9 Binding of B cell CREB/ ATF to a radiolabelled doublestranded CRE probe (CREB)
  • Whole cell extracts from CH12.LX B cells showed 2 retarded bands when analyzed by EMSA with the CRE probe (free probe is off the bottom of the figure). The CREB/ATF protein(s) binding to the CRE were competed by the indicated amount of cold CRE, and by single-stranded CpG ODN, but not by non-CpG ODN.
    • Equivalents
  • Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
    • SEQUENCE LISTING
    • (1) GENERAL INFORMATION:
      • (i) APPLICANT: Arthur M. Krieg, M.D.
      • (ii) TITLE OF INVENTION: IMMUNOMODUIATORY OLIGONUCLEOTIDES
      • (iii) NUMBER OF SEQUENCES: 27
      • (iv) CORRESPONDENCE ADDRESS:
        1. (A) ADDRESSEE: LAHIVE & COCKFIELD
        2. (B) STREET: 60 STATE STREET, SUITE 510
        3. (C) CITY: BOSTON
        4. (D) STATE: MASSACHUSETTS
        5. (E) COUNTRY: USA
        6. (F) ZIP: 02109-1875
      • (v) COMPUTER READABLE FORM:
        1. (A) MEDIUM TYPE: Floppy disk
        2. (B) COMPUTER: IBM PC compatible
        3. (C) OPERATING SYSTEM: PC-DOS/MS-DOS
        4. (D) SOFTWARE: ASCII text
      • (vi) CURRENT APPLICATION DATA:
        1. (A) APPLICATION NUMBER: US
        2. (B) FILING DATE:
        3. (C) CLASSIFICATION:
      • (viii) ATTORNEY/AGENT INFORMATION:
        1. (A) NAME: ARNOLD, BETH B.
        2. (B) REGISTRATION NUMBER: 35,430
        3. (C) REFERENCE/DOCKET NUMBER: UIZ-013CP
      • (ix) TELECOMMUNICATION INFORMATION:
        1. (A) TELEPHONE: (617)227-7400
        2. (B) TELEFAX: (617)227-5941
    • (2) INFORMATION FOR SEQ ID NO: 1:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
        GGGGTCAACG TTCAGGGGGG    20
    • (2) INFORMATION FOR SEQ ID NO:2:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 15 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEMOSS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
        GCTAGACGTT AGCGT    15
    • (2) INFORMATION FOR SEQ ID NO:3:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 15 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
        GCTAGATGTT AGCGT    15
    • (2) INFORMATION FOR SEQ ID NO:4:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 15 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (ix) FEATURE:
        • (A) NAME/KEY: misc_feature
        • (B) LOCATION: 7
        • (D) OTHER INFORMATION: "N indicates 5 methyl cytosine"
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
        GCTAGANGTT AGCGT    15
    • (2) INFORMATION FOR SEQ ID NO:5:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 15 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (ix) FEATURE:
        • (A) NAME/KEY: misc_feature
        • (B) LOCATION:    13
        • (D) OTHER INFORMATION: "N indicates 5 methyl cytosine"
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
        GCTAGACGTT AGNGT    15
    • (2) INFORMATION FOR SEQ ID NO:6:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 15 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
        GCATGACGTT GAGCT    15
    • (2) INFORMATION FOR SEQ ID NO:7:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
        ATGGAAGGTC CAGCGTTCTC    20
    • (2) INFORMATION FOR SEQ ID NO:8:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDECNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (xi) SEQUENCE DESCRIPTION: SBQ ID NO:8:
        ATCGACTCTC GAGCGTTCTC    20
    • (2) INFORMATION FOR SEQ ID NO:9:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (ix) FEATURE:
        • (A) NAME/KEY: misc_feature
        • (B) LOCATION: 3
        • (D) OTHER INFORMATION: "N indicates 5 methyl cytosine"
      • (ix) FEATURE:
        • (A) NAME/KEY: misc_feature
        • (B) LOCATION: 10
        • (D) OTHER INFORMATION: "N indicates 5 methyl cytosine"
      • (ix) FEATURE:
        • (A) NAME/KEY: misc_feature
        • (B) LOCATION: 14
        • (D) OTHER INFORMATION: "N indicates 5 methyl cytosine"
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
        ATNGACTCTN GAGNGTTCTC    20
    • (2) INFORMATION FOR SEQ ID NO:10:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (ix) FEATURE:
        • (A) NAME/KEY: misc_feature
        • (B) LOCATION: 3
        • (D) OTHER INFORMATION: "N indicates 5 methyl cytosine"
      • (xi) SEQUENCE DESCRIPTION: SBQ ID NO:10:
        ATNGACTCTC GAGCGTTCTC    20
    • (2) INFORMATION FOR SBQ ID NO:11:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (ix) FEATURE:
        • (A) NAME/KEY: misc_feature
        • (B) LOCATION: 18
        • (D) OTHER INFORMATION: "N indicates 5 methyl cytosine"
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
        ATCGACTCTC GAGCGTTNTC    20
    • (2) INFORMATION FOR SEQ ID NO:12:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
        ATGGAAGGTC CAACGTTCTC    20
    • (2) INFORMATION FOR SEQ ID NO:13:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
        GAGAACGCTG GACCTTCCAT    20
    • (2) INFORMATION FOR SEQ ID NO:14:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
        GAGAACGCTC GACCTTCCAT    20
    • (2) INFORMATION FOR SEQ ID NO:15:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
        GAGAACGCTC GACCTTCGAT    20
    • (2) INFORMATION FOR SEQ ID NO:16:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (xi) SEQUENCE DESCRIPTION: SBQ ID NO:16:
        GAGCAAGCTG GACCTTCCAT    20
    • (2) INFORMATION FOR SEQ ID NO:17:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (ix) FEATURE:
        • (A) NAME/KEY: mise_feature
        • (B) LOCATION: 6
        • (D) OTHER INFORMATION: "N indicates 5 methyl cytosine"
      • (xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
        QAOAANGCTG GACCTTCCAT    20
    • (2) INFORMATION FOR SBQ ID N0:18:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (ix) FEATURE:
        • (A) NAME/KEY: misc_feature
        • (B) LOCATION: 14
        • (D) OTHER INFORMATION: "N indicates 5 methyl cytosine"
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
        GAGAACGCTG GACNTTCCAT    20
    • (2) INFORMATION FOR SEQ ID NO:19:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
        GAGAACGATG GACCTTCCAT    20
    • (2) INFORMATION FOR SEQ ID NO:20:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH : 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
        GAGAACGCTC CAGCACTGAT    20
    • (2) INFORMATION FOR SEQ ID NO:21:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
        TCCATGTCGG TCCTGATGCT    20
    • (2) INFORMATION FOR SEQ ID NO:22:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
        TCCATGCTGG TCCTGATGCT    20
    • (2) INFORMATION FOR SEQ ID NO:23:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (ix) FEATURE:
        • (A) NAME/KEY: misc_feature
        • (B) LOCATION: 8
        • (D) OTHER INFORMATION: "N indicates 5 methyl cytosine"
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
        TCCATGTNGG TCCTGATGCT    20
    • (2) INFORMATION FOR SEQ ID NO:24:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (ix) FEATURE:
        • (A) NAME/KEY: misc_feature
        • (B) LOCATION: 12
        • (D) OTHER INFORMATION: "N indicates 5 methyl cytosine"
      • (xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
        TCCATGTCGG TNCTGATGCT    20
    • (2) INFORMATION FOR SBQ ID N0:25:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
        TCCATGACGT TCCTGATGCT    20
    • (2) INFORMATION FOR SEQ ID NO:26:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDMMMSS: single
        4. (D) TOPOL40W: linear
      • (ii) MOLECULE TYPE: DNA
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
        TCCATGTCGG TCCTGCTGAT    20
    • (2) INFORMATION FOR SEQ ID NO:27:
      • (i) SEQUENCE CHARACTERISTICS:
        1. (A) LENGTH: 20 base pairs
        2. (B) TYPE: nucleic acid
        3. (C) STRANDEDNESS: single
        4. (D) TOPOLOGY: linear
      • (ii) MOLECULE TYPE: DNA
      • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
        GGGGTCAAGT CTGAGGGGGG    20

Claims (34)

  1. A composition, for use as a medicament, comprising a vaccine and a synthetic immunostimulatory oligonucleotide of at least 8 bases in size, which contains at least one unmethylated CpG dinucleotide and is an adjuvant for the vaccine.
  2. A composition as claimed in claim 1, wherein the immunostimulatory oligonucleotide includes no more than 100 nucleotides.
  3. A composition as claimed in claim 1 or 2, wherein the immunostimulatory oligonucleotide is a phosphodiester.
  4. A composition as claimed in claim 1 or 2, wherein the immunostimulatory oligonucleotide is stabilised.
  5. A composition as claimed in claim 4, wherein the immunostimulatory oligonucleotide has a phosphate backbone modification.
  6. A composition as claimed in claim 5, wherein the modification is a phosphorothioate backbone modification.
  7. A composition as claimed in any one of claims 1-6, wherein the immunostimulatory oligonucleotide is in the range of 8-40 bases in size.
  8. A composition as claimed in any one of claims 1-7, wherein the immunostimulatory oligonucleotide includes more than 8 nucleotides, but does not contain a six base palindrome.
  9. A composition as claimed in any one of claims 1-8, wherein the immunostimulatory oligonucleotide has a mitogenic effect on vertebrate lymphocytes.
  10. A composition as claimed in any one of claims 1-9, wherein the immunostimulatory oligonucleotide contains up to 100 nucleotides and a sequence represented by the following formula:

            5' X1X2CGX3X4 3'

    wherein C and G are unmethylated and X1, X2, X3 and X4 are nucleotides and a G C G trinucleotide sequence is not present at the 5' and 3' termini.
  11. A composition as claimed in claim 10, wherein X1, X2 is a GpA dinucleotide.
  12. A composition as claimed in claim 10 or 11, wherein X3, X4 is a TpC or TpT dinucleotide.
  13. A composition as claimed in claim 10, 11 or 12, wherein the sequence is represented by the following formula:

            5' TX1X2CGX3X4 3'

    wherein C and G are unmethylated and X1, X2, X3 and X4 are nucleotides.
  14. A composition as claimed in any one of claims 1-13, wherein the immunostimulatory oligonucleotide is in the form of an oligonucleotide delivery complex.
  15. A composition as claimed in claim 14, wherein the immunostimulatory oligonucleotide is associated with a sterol, a lipid, or a target cell specific binding agent.
  16. A composition as claimed in claim 15, wherein the immunostimulatory oligonucleotide is associated with cholesterol, a cationic lipid, a virosome, a liposome or a ligand recognised by a target cell specific receptor.
  17. A composition as claimed in claim 15, wherein the target cell specific binding agent is specific for a B-cell.
  18. A composition as claimed in any one of claims 1-17, wherein the immunostimulatory oligonucleotide includes a plurality of unmethylated CpG dinucleotides.
  19. A composition as claimed in any one of claims 1-18, further comprising a pharmaceutically acceptable carrier.
  20. A composition as claimed in any one of the preceding claims, for use in treating:-
    (a) a human subject; or
    (b) a non-human vertebrate animal, optionally, a dog, cat, horse, cow, pig, sheep, goat, chicken, monkey, rat or mouse.
  21. A composition as claimed in any one of claims 1-20, for treating, preventing or ameliorating a disease or disorder in which a subject's immune system is not functioning normally, or in which it would be useful to boost a subject's immune response.
  22. A composition as claimed in claim 21, wherein said disease or disorder is a tumour or cancer, or a viral, fungal, bacterial or parasitic infection.
  23. Use of a composition as claimed in any one of claims 1-19, for the manufacture of a medicament for vaccinating a subject.
  24. Use of a synthetic immunostimulatory oligonucleotide of at least 8 bases in size, which contains at least one unmethylated CpG dinucleotide, for the manufacture of a medicament for administration to a subject in conjunction with a vaccine, as an adjuvant for the vaccine.
  25. A use as claimed in claim 24, wherein the immunostimulatory oligonucleotide is an immunostimulatory oligonucleotide as defined in any one of claims 2-18.
  26. A use as claimed in claim 24 or 25, wherein said immunostimulatory oligonucleotide and said vaccine are for use in a method of treating, preventing or ameliorating a disease or disorder in which a subject's immune system is not functioning normally, or in which it would be useful to boost a subject's immune response.
  27. A use as claimed in any one of claim 23-26, wherein the medicament boosts an immune response to a tumour or a cancer, or a viral, fungal, bacterial or parasitic infection.
  28. Use of a composition as claimed in any one of claims 1-19 for the manufacture of a medicament for use in a method of treating, preventing or ameliorating a disease or disorder in which a subject's immune system is not functioning normally, or in which it would be useful to boost a subject's immune response.
  29. A use as claimed in claim 26 or 28, wherein said disease or disorder is a tumor or cancer, or a viral, fungal, bacterial or parasitic infection.
  30. A use as claimed in any one of claims 23-29 wherein the medicament is for activating a subject's B cells.
  31. A use as claimed in claim 23 or 24, wherein the medicament is for use in treating, preventing, or ameliorating leukaemia.
  32. A use as claimed in any one of claims 23-31, wherein the composition is formulated for oral, transdermal, subcutaneous, intravenous, parenteral, interperitoneal, or intrathecal administration.
  33. Use of a composition as claimed in any one of claims 1-19, for the manufacture of a medicament for treating:-
    (a) a human subject; or,
    (b) a non-human vertebrate animal, optionally, a dog, cat, horse, cow, pig, sheep, goat, chicken, monkey, rat or mouse.
  34. A use as claimed in any one of claims 23-32, wherein the medicament is for treating:-
    (a) a human subject; or,
    (b) a non-human vertebrate animal, optionally, a dog, cat, horse, cow, pig, sheep, goat, chicken, monkey, rat or mouse.
EP95911630A 1994-07-15 1995-02-07 Immunomodulatory oligonucleotides Expired - Lifetime EP0772619B2 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP01202814A EP1167379A3 (en) 1994-07-15 1995-02-07 Immunomodulatory oligonucleotides
DE69535036T DE69535036T3 (en) 1994-07-15 1995-02-07 IMMUNOMODULATIVE OLIGONUCLEOTIDES
EP01202811A EP1167377B2 (en) 1994-07-15 1995-02-07 Immunomodulatory oligonucleotides
EP01202813A EP1167378B1 (en) 1994-07-15 1995-02-07 Immunomodulatory oligonucleotides

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US27635894A 1994-07-15 1994-07-15
US276358 1994-07-15
PCT/US1995/001570 WO1996002555A1 (en) 1994-07-15 1995-02-07 Immunomodulatory oligonucleotides

Related Child Applications (10)

Application Number Title Priority Date Filing Date
EP01202811A Division EP1167377B2 (en) 1994-07-15 1995-02-07 Immunomodulatory oligonucleotides
EP01202814A Division EP1167379A3 (en) 1994-07-15 1995-02-07 Immunomodulatory oligonucleotides
EP01202813A Division EP1167378B1 (en) 1994-07-15 1995-02-07 Immunomodulatory oligonucleotides
EP01202814.8 Division-Into 2001-07-23
EP01202813.0 Division-Into 2001-07-23
EP01202811.4 Division-Into 2001-07-23
EP04026517.5 Division-Into 2004-11-09
EP04105677.1 Division-Into 2004-11-10
EP08100614.0 Division-Into 2008-01-17
EP08100615.7 Division-Into 2008-01-17

Publications (3)

Publication Number Publication Date
EP0772619A1 EP0772619A1 (en) 1997-05-14
EP0772619B1 EP0772619B1 (en) 2006-06-07
EP0772619B2 true EP0772619B2 (en) 2010-12-08

Family

ID=23056334

Family Applications (4)

Application Number Title Priority Date Filing Date
EP01202814A Ceased EP1167379A3 (en) 1994-07-15 1995-02-07 Immunomodulatory oligonucleotides
EP01202811A Expired - Lifetime EP1167377B2 (en) 1994-07-15 1995-02-07 Immunomodulatory oligonucleotides
EP01202813A Expired - Lifetime EP1167378B1 (en) 1994-07-15 1995-02-07 Immunomodulatory oligonucleotides
EP95911630A Expired - Lifetime EP0772619B2 (en) 1994-07-15 1995-02-07 Immunomodulatory oligonucleotides

Family Applications Before (3)

Application Number Title Priority Date Filing Date
EP01202814A Ceased EP1167379A3 (en) 1994-07-15 1995-02-07 Immunomodulatory oligonucleotides
EP01202811A Expired - Lifetime EP1167377B2 (en) 1994-07-15 1995-02-07 Immunomodulatory oligonucleotides
EP01202813A Expired - Lifetime EP1167378B1 (en) 1994-07-15 1995-02-07 Immunomodulatory oligonucleotides

Country Status (12)

Country Link
US (1) US6194388B1 (en)
EP (4) EP1167379A3 (en)
JP (7) JP3468773B2 (en)
AT (3) ATE328890T1 (en)
AU (1) AU713040B2 (en)
CA (2) CA2194761C (en)
DE (2) DE69535036T3 (en)
DK (1) DK0772619T4 (en)
ES (3) ES2320315T5 (en)
HK (3) HK1043598A1 (en)
PT (1) PT772619E (en)
WO (1) WO1996002555A1 (en)

Families Citing this family (844)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5849719A (en) 1993-08-26 1998-12-15 The Regents Of The University Of California Method for treating allergic lung disease
US6727230B1 (en) * 1994-03-25 2004-04-27 Coley Pharmaceutical Group, Inc. Immune stimulation by phosphorothioate oligonucleotide analogs
ATE328890T1 (en) * 1994-07-15 2006-06-15 Univ Iowa Res Found IMMUNOMODULATORY OLIGONUCLEOTIDES
US7935675B1 (en) * 1994-07-15 2011-05-03 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6429199B1 (en) 1994-07-15 2002-08-06 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules for activating dendritic cells
US20030026782A1 (en) * 1995-02-07 2003-02-06 Arthur M. Krieg Immunomodulatory oligonucleotides
US6239116B1 (en) 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6207646B1 (en) * 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US7422902B1 (en) * 1995-06-07 2008-09-09 The University Of British Columbia Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer
US5981501A (en) * 1995-06-07 1999-11-09 Inex Pharmaceuticals Corp. Methods for encapsulating plasmids in lipid bilayers
DE69737413T2 (en) 1996-01-04 2007-10-31 Novartis Vaccines and Diagnostics, Inc., Emeryville BACTERIOFERRITINE FROM HELICOBACTER PYLORI
ATE437943T1 (en) * 1996-01-30 2009-08-15 Univ California EXPRESSION VECTORS THAT INDUCE AN ANTIGEN-SPECIFIC IMMUNE RESPONSE AND METHODS FOR THEIR USE.
FR2750704B1 (en) * 1996-07-04 1998-09-25 Rhone Poulenc Rorer Sa PROCESS FOR PRODUCING THERAPEUTIC DNA
US5856462A (en) * 1996-09-10 1999-01-05 Hybridon Incorporated Oligonucleotides having modified CpG dinucleosides
AU768178B2 (en) * 1996-10-11 2003-12-04 Regents Of The University Of California, The Immunostimulatory polynucleotide/immunomodulatory molecule conjugates
EP0930893B1 (en) 1996-10-11 2005-04-13 The Regents of The University of California Immunostimulatory polynucleotide/immunomodulatory molecule conjugates
EP0855184A1 (en) * 1997-01-23 1998-07-29 Grayson B. Dr. Lipford Pharmaceutical composition comprising a polynucleotide and an antigen especially for vaccination
EP1039935A4 (en) * 1997-02-28 2005-04-27 Univ Iowa Res Found USE OF NUCLEIC ACIDS CONTAINING UNMETHYLATED CpG DINUCLEOTIDE IN THE TREATMENT OF LPS-ASSOCIATED DISORDERS
US6406705B1 (en) * 1997-03-10 2002-06-18 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
WO1998040100A1 (en) * 1997-03-10 1998-09-17 Ottawa Civic Loeb Research Institute USE OF NUCLEIC ACIDS CONTAINING UNMETHYLATED CpG DINUCLEOTIDE AS AN ADJUVANT
US6426334B1 (en) 1997-04-30 2002-07-30 Hybridon, Inc. Oligonucleotide mediated specific cytokine induction and reduction of tumor growth in a mammal
US20030104044A1 (en) * 1997-05-14 2003-06-05 Semple Sean C. Compositions for stimulating cytokine secretion and inducing an immune response
DE69841002D1 (en) 1997-05-14 2009-09-03 Univ British Columbia Highly effective encapsulation of nucleic acids in lipid vesicles
JP2001526688A (en) * 1997-05-19 2001-12-18 メルク エンド カンパニー インコーポレーテッド Oligonucleotide adjuvant
WO1998052581A1 (en) 1997-05-20 1998-11-26 Ottawa Civic Hospital Loeb Research Institute Vectors and methods for immunization or therapeutic protocols
DE69840850D1 (en) * 1997-06-06 2009-07-09 Dynavax Tech Corp INHIBITORS OF IMMUNSTIMULATORY DNA SEQUENCE ACTIVITY
EP1374894A3 (en) * 1997-06-06 2004-09-22 Dynavax Technologies Corporation Immunostimulatory oligonucleotides, compositions thereof and methods of use thereof
US6589940B1 (en) 1997-06-06 2003-07-08 Dynavax Technologies Corporation Immunostimulatory oligonucleotides, compositions thereof and methods of use thereof
US20040006034A1 (en) * 1998-06-05 2004-01-08 Eyal Raz Immunostimulatory oligonucleotides, compositions thereof and methods of use thereof
ES2283070T3 (en) * 1997-09-05 2007-10-16 The Regents Of The University Of California USE OF IMMUNOSTIMULANT OLIGONUCLEOTIDES FOR THE PREVENTION OR TREATMENT OF ASTHMA.
GB9724531D0 (en) 1997-11-19 1998-01-21 Smithkline Biolog Novel compounds
EP1584685B1 (en) 1998-02-05 2011-04-13 GlaxoSmithKline Biologicals SA Tumor-associated antigen derivatives from the mage family, used for the preparation of fusion proteins with T-helper epitopes and of compositions for vaccination
US20020147143A1 (en) 1998-03-18 2002-10-10 Corixa Corporation Compositions and methods for the therapy and diagnosis of lung cancer
AU760549B2 (en) 1998-04-03 2003-05-15 University Of Iowa Research Foundation, The Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines
IL138000A0 (en) * 1998-04-09 2001-10-31 Smithkline Beecham Biolog Adjuvant compositions
GB9808866D0 (en) 1998-04-24 1998-06-24 Smithkline Beecham Biolog Novel compounds
EP1077708A1 (en) * 1998-05-06 2001-02-28 University Of Iowa Research Foundation Methods for the prevention and treatment of parasitic infections and related diseases using cpg oligonucleotides
IL139646A0 (en) * 1998-05-14 2002-02-10 Coley Pharm Group Inc Methods for regulating hematopoiesis using cpg-oligonucleotides
RU2244547C2 (en) 1998-05-19 2005-01-20 Рисерч Дивелопмент Фаундейшн Triterpene compositions and methods for their applying
EP1077722B1 (en) * 1998-05-22 2006-08-09 Ottawa Health Research Institute Methods and products for inducing mucosal immunity
US6881561B1 (en) 1998-05-27 2005-04-19 Cheil Jedang Corporation Endonuclease of immune cell, process for producing the same and immune adjuvant using the same
KR19990086271A (en) * 1998-05-27 1999-12-15 손경식 Novel endonucleases of immune cells and immunoadjuvant using the same
US6562798B1 (en) * 1998-06-05 2003-05-13 Dynavax Technologies Corp. Immunostimulatory oligonucleotides with modified bases and methods of use thereof
US20030022854A1 (en) 1998-06-25 2003-01-30 Dow Steven W. Vaccines using nucleic acid-lipid complexes
US20040247662A1 (en) * 1998-06-25 2004-12-09 Dow Steven W. Systemic immune activation method using nucleic acid-lipid complexes
US6693086B1 (en) * 1998-06-25 2004-02-17 National Jewish Medical And Research Center Systemic immune activation method using nucleic acid-lipid complexes
WO2000006588A1 (en) * 1998-07-27 2000-02-10 University Of Iowa Research Foundation STEREOISOMERS OF CpG OLIGONUCLEOTIDES AND RELATED METHODS
CN102198266A (en) 1998-07-31 2011-09-28 株式会社国际癌症免疫研究所 Cancer antigens based on tumor suppressor gene WT1 product
PL346213A1 (en) 1998-08-07 2002-01-28 Univ Washington Immunological herpes simplex virus antigens and methods for use thereof
DK1104306T3 (en) * 1998-08-10 2006-05-22 Antigenics Inc Preparations of CpG and Saponin Adjuvants and Methods for Using Them
EP1109924A2 (en) * 1998-09-09 2001-06-27 Genzyme Corporation Methylation of plasmid vectors
US6692752B1 (en) 1999-09-08 2004-02-17 Smithkline Beecham Biologicals S.A. Methods of treating human females susceptible to HSV infection
FR2783170B1 (en) * 1998-09-11 2004-07-16 Pasteur Merieux Serums Vacc IMMUNOSTIMULATING EMULSION
ATE326239T1 (en) * 1998-09-18 2006-06-15 Dynavax Tech Corp METHODS FOR TREATING IG-E ASSOCIATED DISEASES AND COMPOSITIONS FOR USE IN SUCH METHODS
US20030235557A1 (en) 1998-09-30 2003-12-25 Corixa Corporation Compositions and methods for WT1 specific immunotherapy
WO2000020039A1 (en) * 1998-10-05 2000-04-13 The Regents Of The University Of California Methods and adjuvants for stimulating mucosal immunity
CN100558401C (en) 1998-10-16 2009-11-11 史密丝克莱恩比彻姆生物有限公司 Adjuvant system and vaccine
CA2354028A1 (en) 1998-12-08 2000-06-15 Smithkline Beecham Biologicals S.A. Neisseria meningitidis basb041 polypeptides and encoding polynucleotides and uses thereof
ATE469222T1 (en) 1998-12-08 2010-06-15 Corixa Corp COMPOUNDS AND METHODS FOR THE TREATMENT AND DIAGNOSIS OF CHLAMYDIA INFECTIONS
US20020119158A1 (en) 1998-12-17 2002-08-29 Corixa Corporation Compositions and methods for the therapy and diagnosis of ovarian cancer
US6579973B1 (en) 1998-12-28 2003-06-17 Corixa Corporation Compositions for the treatment and diagnosis of breast cancer and methods for their use
US7198920B1 (en) * 1999-01-29 2007-04-03 Corika Corporation HER-2/neu fusion proteins
CA2361459A1 (en) * 1999-02-05 2000-08-10 Salam Abdul Kadhim Use of cationic lipids to generate anti-tumor immunity
EP2204186B1 (en) 1999-02-17 2016-04-06 CSL Limited Immunogenic complexes and methods relating thereto
ES2228454T3 (en) 1999-02-26 2005-04-16 Chiron S.R.L. IMPROVEMENT OF THE BACTERIAL ACTIVITY OF NEISSERIA ANTIGENS WITH OLIGONUCLEOTIDES CONTAINING MOTIVES CG.
JP2002537102A (en) 1999-02-26 2002-11-05 カイロン コーポレイション Microemulsion with adsorbed polymer and fine particles
ATE451461T1 (en) 1999-03-12 2009-12-15 Glaxosmithkline Biolog Sa ANTIGEN POLYPEPTIDES FROM NEISSERIA MENINGITIDIS, POLYNUCLEOTIDES CODING THEREFOR AND CORRESPONDING PROTECTIVE ANTIBODIES
JP4846906B2 (en) 1999-03-19 2011-12-28 グラクソスミスクライン バイオロジカルズ ソシエテ アノニム vaccine
JP2000262247A (en) * 1999-03-19 2000-09-26 Asama Kasei Kk Immunomodulation agent, immunomodulation food and immunomodulation feed
GB9909077D0 (en) 1999-04-20 1999-06-16 Smithkline Beecham Biolog Novel compositions
FR2790955B1 (en) * 1999-03-19 2003-01-17 Assist Publ Hopitaux De Paris USE OF STABILIZED OLIGONUCLEOTIDES AS ANTI-TUMOR ACTIVE INGREDIENT
NZ514818A (en) 1999-04-02 2004-04-30 Corixa Corp Compounds and methods for therapy and diagnosis of lung cancer
WO2000061151A2 (en) * 1999-04-12 2000-10-19 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Oligodeoxynucleotide and its use to induce an immune response
US6977245B2 (en) 1999-04-12 2005-12-20 The United States Of America As Represented By The Department Of Health And Human Services Oligodeoxynucleotide and its use to induce an immune response
IL145982A0 (en) 1999-04-19 2002-07-25 Smithkline Beecham Biolog Vaccines
US6558670B1 (en) 1999-04-19 2003-05-06 Smithkline Beechman Biologicals S.A. Vaccine adjuvants
DE60013591T2 (en) * 1999-04-29 2005-02-03 Coley Pharmaceutical Gmbh SCREENING FOR MODULATORS OF THE FUNCTION OF IMMUNSTIMULATORY DNA
CA2376992A1 (en) * 1999-06-29 2001-01-04 Smithkline Beecham Biologicals S.A. Use of cpg as an adjuvant for hiv vaccine
GB9915204D0 (en) * 1999-06-29 1999-09-01 Smithkline Beecham Biolog Vaccine
US6514948B1 (en) 1999-07-02 2003-02-04 The Regents Of The University Of California Method for enhancing an immune response
FR2795963A1 (en) * 1999-07-08 2001-01-12 Pasteur Merieux Serums Vacc New polynucleotides are useful as vaccines for humans
SK782002A3 (en) * 1999-07-21 2003-08-05 Lexigen Pharm Corp FC fusion proteins for enhancing the immunogenicity of protein and peptide antigens
GB9918319D0 (en) 1999-08-03 1999-10-06 Smithkline Beecham Biolog Vaccine composition
US20050226890A1 (en) * 1999-08-12 2005-10-13 Cohen David I Tat-based vaccine compositions and methods of making and using same
US20050244434A1 (en) * 1999-08-12 2005-11-03 Cohen David I Tat-based tolerogen compositions and methods of making and using same
CA2380947C (en) 1999-08-19 2011-11-01 Dynavax Technologies Corporation Methods of modulating an immune response using immunostimulatory sequences and compositions for use therein
MXPA02002107A (en) * 1999-08-27 2003-08-20 Inex Pharmaceuticals Corp Compositions for stimulating cytokine secretion and inducing an immune response.
NZ517929A (en) * 1999-09-25 2004-02-27 Univ Iowa Res Found Immunostimulatory nucleic acids
US6949520B1 (en) 1999-09-27 2005-09-27 Coley Pharmaceutical Group, Inc. Methods related to immunostimulatory nucleic acid-induced interferon
CA2388337C (en) 1999-10-22 2013-01-08 Aventis Pasteur Limited Method of inducing and/or enhancing an immune response to tumor antigens
US7223398B1 (en) 1999-11-15 2007-05-29 Dynavax Technologies Corporation Immunomodulatory compositions containing an immunostimulatory sequence linked to antigen and methods of use thereof
WO2001045750A1 (en) * 1999-12-21 2001-06-28 The Regents Of The University Of California Method for preventing an anaphylactic reaction
AU2001227889A1 (en) 2000-01-14 2001-07-24 The United States of America, represented by The Secretary, Department of Health & Human Services Oligodeoxynucleotide and its use to induce an immune response
AT409085B (en) * 2000-01-28 2002-05-27 Cistem Biotechnologies Gmbh PHARMACEUTICAL COMPOSITION FOR IMMUNULATING AND PRODUCING VACCINES
EP1253947A4 (en) * 2000-01-31 2005-01-05 Univ California Immunomodulatory polynucleotides in treatment of an infection by an intracellular pathogen
US7585847B2 (en) * 2000-02-03 2009-09-08 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids for the treatment of asthma and allergy
FR2805264B1 (en) * 2000-02-18 2002-04-12 Aventis Pasteur IMMUNOSTIMULATING OLIGONUCLEOTIDES
FR2805265B1 (en) * 2000-02-18 2002-04-12 Aventis Pasteur IMMUNOSTIMULATING OLIGONUCLEOTIDES
WO2001062207A2 (en) * 2000-02-23 2001-08-30 The Regents Of The University Of California Method for treating inflammatory bowel disease and other forms of gastrointestinal inflammation
PL209127B1 (en) * 2000-02-23 2011-07-29 Smithkline Beecham Biolog Tumour-specific animal proteins
US20030130217A1 (en) * 2000-02-23 2003-07-10 Eyal Raz Method for treating inflammatory bowel disease and other forms of gastrointestinal inflammation
US20040002068A1 (en) 2000-03-01 2004-01-01 Corixa Corporation Compositions and methods for the detection, diagnosis and therapy of hematological malignancies
US20040131628A1 (en) * 2000-03-08 2004-07-08 Bratzler Robert L. Nucleic acids for the treatment of disorders associated with microorganisms
US7157437B2 (en) 2000-03-10 2007-01-02 Dynavax Technologies Corporation Methods of ameliorating symptoms of herpes infection using immunomodulatory polynucleotide sequences
US20020098199A1 (en) 2000-03-10 2002-07-25 Gary Van Nest Methods of suppressing hepatitis virus infection using immunomodulatory polynucleotide sequences
US20010046967A1 (en) 2000-03-10 2001-11-29 Gary Van Nest Methods of preventing and treating respiratory viral infection using immunomodulatory polynucleotide
US7129222B2 (en) 2000-03-10 2006-10-31 Dynavax Technologies Corporation Immunomodulatory formulations and methods for use thereof
US20030129251A1 (en) 2000-03-10 2003-07-10 Gary Van Nest Biodegradable immunomodulatory formulations and methods for use thereof
US20020142978A1 (en) * 2000-04-07 2002-10-03 Eyal Raz Synergistic improvements to polynucleotide vaccines
EP1278855B1 (en) 2000-04-21 2008-03-12 Corixa Corporation Compounds and methods for treatment and diagnosis of chlamydial infection
ES2276788T3 (en) 2000-05-10 2007-07-01 Sanofi Pasteur Limited IMMUNOGEN POLIPEPTIDES CODED BY MAGE MINIGENS AND THEIR USES.
DE60134134D1 (en) 2000-05-19 2008-07-03 Corixa Corp PROPHYLACTIC AND THERAPEUTIC TREATMENT OF TEN WITH COMPOUNDS BASED ON MONOSACCHARIDES
JP5139618B2 (en) 2000-06-20 2013-02-06 コリクサ コーポレイション Mycobacterium tuberculosis fusion protein
DK1296714T3 (en) 2000-06-22 2009-12-07 Coley Pharm Gmbh Combination of CpG and antibodies directed against CD19, CD20, CD22 or CD40 for the treatment or prevention of cancer
ATE396265T1 (en) 2000-06-28 2008-06-15 Corixa Corp COMPOSITIONS AND METHODS FOR THERAPY AND DIAGNOSIS OF LUNG CANCER
GB0108364D0 (en) 2001-04-03 2001-05-23 Glaxosmithkline Biolog Sa Vaccine composition
ES2385100T3 (en) 2000-06-29 2012-07-18 Smithkline Beecham Biologicals S.A. Composition of multivalent vaccine with reduced dose of Haemophilus influenzae type B
US7229623B1 (en) 2000-08-03 2007-06-12 Corixa Corporation Her-2/neu fusion proteins
KR100917101B1 (en) * 2000-08-04 2009-09-15 도요 보세키 가부시키가이샤 Flexible metal laminate and production method thereof
UA79735C2 (en) 2000-08-10 2007-07-25 Глаксосмітклайн Байолоджікалз С.А. Purification of hbv antigens for use in vaccines
WO2002011761A2 (en) * 2000-08-10 2002-02-14 Henry M. Jackson Foundation For The Advancement Of Military Medicine Vaccine against rsv
EP1335741A4 (en) * 2000-08-25 2005-10-26 Yeda Res & Dev METHODS OF TREATMENT OR PREVENTION OF AUTOIMMUNE DISEASES WITH CpG-CONTAINING POLYNUCLEOTIDE
GB0022742D0 (en) 2000-09-15 2000-11-01 Smithkline Beecham Biolog Vaccine
EP1366077B1 (en) * 2000-09-15 2011-05-25 Coley Pharmaceutical GmbH PROCESS FOR HIGH THROUGHPUT SCREENING OF CpG-BASED IMMUNO-AGONIST/ANTAGONIST
DK2266603T3 (en) 2000-10-18 2012-11-05 Glaxosmithkline Biolog Sa tumor Vaccines
AU1412702A (en) 2000-10-27 2002-05-06 Chiron Spa Nucleic acids and proteins from streptococcus groups a and b
CA2598144A1 (en) * 2000-12-08 2006-08-31 3M Innovative Properties Company Compositions and methods for targeted delivery of immune response modifiers
US6677347B2 (en) * 2000-12-08 2004-01-13 3M Innovative Properties Company Sulfonamido ether substituted imidazoquinolines
AU2001297693A1 (en) * 2000-12-08 2002-09-12 Coley Pharmaceutical Gmbh Cpg-like nucleic acids and methods of use thereof
CA2430691A1 (en) 2000-12-27 2002-07-04 Dynavax Technologies Corporation Immunomodulatory polynucleotides and methods of using the same
CA2434573A1 (en) * 2001-01-12 2002-07-18 Amato Pharmaceutical Products, Ltd. Agent for protection against microorganism infection
CN1496266A (en) * 2001-01-12 2004-05-12 ������ҩ��ʽ���� Antiallergic agents
US7128911B2 (en) 2001-01-19 2006-10-31 Cytos Biotechnology Ag Antigen arrays for treatment of bone disease
CA2436000A1 (en) * 2001-01-24 2002-08-08 Susumu Okabe An antistress agent
DE10105234A1 (en) 2001-02-02 2002-08-29 Schoeller Textil Ag Sevelen Textile surface
GB0103171D0 (en) 2001-02-08 2001-03-28 Smithkline Beecham Biolog Vaccine composition
GB0105360D0 (en) * 2001-03-03 2001-04-18 Glaxo Group Ltd Chimaeric immunogens
US20040097703A1 (en) 2001-03-22 2004-05-20 Haruo Sugiyama Wt1 modified peptide
US20030050268A1 (en) * 2001-03-29 2003-03-13 Krieg Arthur M. Immunostimulatory nucleic acid for treatment of non-allergic inflammatory diseases
EP1988097A1 (en) 2001-05-09 2008-11-05 Corixa Corporation Compositions and methods for the therapy and diagnosis of prostate cancer
US20050070462A1 (en) 2001-05-21 2005-03-31 Intercell Ag Method for stabilising of nucleic acids
US6818787B2 (en) * 2001-06-11 2004-11-16 Xenoport, Inc. Prodrugs of GABA analogs, compositions and uses thereof
GB0115176D0 (en) 2001-06-20 2001-08-15 Chiron Spa Capular polysaccharide solubilisation and combination vaccines
US7785610B2 (en) * 2001-06-21 2010-08-31 Dynavax Technologies Corporation Chimeric immunomodulatory compounds and methods of using the same—III
PT1404873E (en) * 2001-06-21 2013-07-30 Dynavax Tech Corp Chimeric immunomodulatory compounds and methods of using the same
CZ20033515A3 (en) * 2001-06-29 2005-01-12 Chiron Corporation HCV E1E2 vaccine composition
CA2452382A1 (en) 2001-07-10 2003-01-23 Corixa Corporation Compositions and methods for delivery of proteins and adjuvants encapsulated in microspheres
GB0118249D0 (en) 2001-07-26 2001-09-19 Chiron Spa Histidine vaccines
US7666674B2 (en) 2001-07-27 2010-02-23 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of sterically stabilized cationic liposomes to efficiently deliver CPG oligonucleotides in vivo
GB0121591D0 (en) 2001-09-06 2001-10-24 Chiron Spa Hybrid and tandem expression of neisserial proteins
US20030148316A1 (en) * 2001-08-01 2003-08-07 Lipford Grayson B. Methods and compositions relating to plasmacytoid dendritic cells
US20030072762A1 (en) * 2001-08-03 2003-04-17 Van De Winkel Jan G. J. Compositions comprising immunostimulatory oligonucleotides and uses thereof to enhance Fc receptor-mediated immunotherapies
AU2002326561B2 (en) * 2001-08-07 2008-04-03 Dynavax Technologies Corporation Immunomodulatory compositions, formulations, and methods for use thereof
AU2002361468A1 (en) * 2001-08-14 2003-03-18 The Government Of The United States Of America As Represented By The Secretary Of Health And Human S Method for rapid generation of mature dendritic cells
US7361352B2 (en) 2001-08-15 2008-04-22 Acambis, Inc. Influenza immunogen and vaccine
CA2457485C (en) 2001-08-17 2012-08-14 Arthur M. Krieg Combination motif immune stimulatory oligonucleotides with improved activity
JP4319908B2 (en) 2001-08-24 2009-08-26 ユニバーシティ オブ ビクトリア イノベーション アンド ディベロップメント コーポレーション Proaerolysin containing a protease activating sequence and methods of use for the treatment of prostate cancer
JP2005501550A (en) * 2001-08-30 2005-01-20 スリーエム イノベイティブ プロパティズ カンパニー Maturation of plasmacytoid dendritic cells using immune response modifier molecules
IL160561A0 (en) * 2001-09-07 2004-07-25 Univ Boston Method and composition for treating immune complex associated disorders
CA2492823A1 (en) * 2001-09-14 2003-03-27 Martin F. Bachmann In vivo activation of antigen presenting cells for enhancement of immune responses induced by virus like particles
US20030099668A1 (en) * 2001-09-14 2003-05-29 Cytos Biotechnology Ag Packaging of immunostimulatory substances into virus-like particles: method of preparation and use
EP2322626A1 (en) 2001-09-20 2011-05-18 Glaxo Group Limited HIV-GAG codon-optimised DNA vaccines
AU2002353783A1 (en) 2001-09-24 2003-04-07 The Government Of The United States Of America As Represented By The Secretary Of The Department Of SUPPRESSORS OF CpG OLIGONUCLEOTIDES AND METHODS OF USE
US20030119774A1 (en) * 2001-09-25 2003-06-26 Marianna Foldvari Compositions and methods for stimulating an immune response
WO2003028758A1 (en) * 2001-09-28 2003-04-10 Haruo Sugiyama Novel method of inducing antigen-specific t cells
WO2003028757A1 (en) * 2001-09-28 2003-04-10 Haruo Sugiyama Novel method of inducing antigen-specific t cells
AR045702A1 (en) 2001-10-03 2005-11-09 Chiron Corp COMPOSITIONS OF ASSISTANTS.
CN1633598A (en) * 2001-10-05 2005-06-29 科勒制药股份公司 Toll-like receptor 3 signaling agonists and antagonists
JP2005519990A (en) * 2001-10-12 2005-07-07 ユニバーシティ オブ アイオワ リサーチ ファウンデーション Methods and products for enhancing immune responses using imidazoquinoline compounds
TW200303759A (en) * 2001-11-27 2003-09-16 Schering Corp Methods for treating cancer
US20050250716A1 (en) * 2001-12-07 2005-11-10 Intercell Ag Immunostimulatory oligodeoxynucleotides
WO2003050241A2 (en) 2001-12-12 2003-06-19 The Government Of The United States Of America As Represented By The Secretary, Department Of Healthand Human Services Methods for using extracellular adenosine inhibitors and adenosine receptor inhibitors to enhance immune response and inflammation
EP1581119B1 (en) 2001-12-17 2013-01-30 Corixa Corporation Compositions and methods for the therapy and diagnosis of inflammatory bowel disease
AU2002366710A1 (en) 2001-12-20 2003-07-09 The Government Of The United States Of America As Represented By The Secretary Of The Department Of USE OF CpG OLIGODEOXYNUCLEOTIDES TO INDUCE ANGIOGENESIS
US8466116B2 (en) 2001-12-20 2013-06-18 The Unites States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of CpG oligodeoxynucleotides to induce epithelial cell growth
US20060210555A1 (en) * 2001-12-21 2006-09-21 Antigenics, Inc. Compositions comprising immunoreactive reagents and saponins, and methods of use thereof
WO2003067286A2 (en) * 2002-02-07 2003-08-14 Covalent Partners, Llc Nanofilm and membrane compositions
US20040034223A1 (en) * 2002-02-07 2004-02-19 Covalent Partners, Llc. Amphiphilic molecular modules and constructs based thereon
US8088388B2 (en) * 2002-02-14 2012-01-03 United Biomedical, Inc. Stabilized synthetic immunogen delivery system
AU2003215316A1 (en) 2002-02-20 2003-09-09 Chiron Corporation Microparticles with adsorbed polypeptide-containing molecules
US7351413B2 (en) 2002-02-21 2008-04-01 Lorantis, Limited Stabilized HBc chimer particles as immunogens for chronic hepatitis
US20070037769A1 (en) * 2003-03-14 2007-02-15 Multicell Immunotherapeutics, Inc. Compositions and methods to treat and control tumors by loading antigen presenting cells
US20050222060A1 (en) * 2002-03-15 2005-10-06 Bot Adrian L Compositions and methods to initiate or enhance antibody and major-histocompatibility class I or class II-restricted t cell responses by using immunomodulatory, non-coding rna motifs
EP2258712A3 (en) 2002-03-15 2011-05-04 Multicell Immunotherapeutics, Inc. Compositions and Methods to Initiate or Enhance Antibody and Major-histocompatibility Class I or Class II-restricted T Cell Responses by Using Immunomodulatory, Non-coding RNA Motifs
RU2302865C2 (en) * 2002-04-04 2007-07-20 Коли Фармасьютикал Гмбх Immunostimulating g,u-containing olygoribonucleotides
US20030224013A1 (en) * 2002-04-19 2003-12-04 Cole Garry T. Methods for protection against Coccidioides spp. infection using Coccidioides spp. urea amidohydrolase (Ure) protein
US20040009943A1 (en) * 2002-05-10 2004-01-15 Inex Pharmaceuticals Corporation Pathogen vaccines and methods for using the same
US20040013649A1 (en) * 2002-05-10 2004-01-22 Inex Pharmaceuticals Corporation Cancer vaccines and methods of using the same
US20040009944A1 (en) * 2002-05-10 2004-01-15 Inex Pharmaceuticals Corporation Methylated immunostimulatory oligonucleotides and methods of using the same
WO2003094963A2 (en) * 2002-05-10 2003-11-20 Inex Pharmaceuticals Corporation Methylated immunostimulatory oligonucleotides and methods of using the same
KR100456681B1 (en) * 2002-05-22 2004-11-10 주식회사 대웅 Immnune-stimulating and controlling Composition comprising bacterial chromosomal DNA fragments and detoxified lipopolysaccharides
CA2388049A1 (en) 2002-05-30 2003-11-30 Immunotech S.A. Immunostimulatory oligonucleotides and uses thereof
SE0201701D0 (en) * 2002-06-05 2002-06-05 Gotovax Ab Treatment of epithelial tumors and infections
AU2003243409A1 (en) * 2002-06-05 2003-12-22 Coley Pharmaceutical Group, Inc. Method for treating autoimmune or inflammatory diseases with combinations of inhibitory oligonucleotides and small molecule antagonists of immunostimulatory cpg nucleic acids
US20040005338A1 (en) * 2002-06-20 2004-01-08 Cytos Biotechnology Ag Packaged virus-like particles for use as adjuvants: method of preparation and use
US7575742B2 (en) 2002-06-28 2009-08-18 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Method of treating autoimmune diseases with interferon-beta and IL-2R antagonist
US7569553B2 (en) * 2002-07-03 2009-08-04 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
DE10229872A1 (en) 2002-07-03 2004-01-29 Curevac Gmbh Immune stimulation through chemically modified RNA
US7605138B2 (en) * 2002-07-03 2009-10-20 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US20040053880A1 (en) * 2002-07-03 2004-03-18 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US7807803B2 (en) * 2002-07-03 2010-10-05 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US7576066B2 (en) * 2002-07-03 2009-08-18 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
WO2004007743A2 (en) * 2002-07-17 2004-01-22 Coley Pharmaceutical Gmbh Use of cpg nucleic acids in prion-disease
ATE494907T1 (en) 2002-07-18 2011-01-15 Univ Washington PHARMACEUTICAL COMPOSITIONS CONTAINING IMMUNOLOGICALLY ACTIVE HERPES SIMPLEX VIRUS PROTEIN FRAGMENTS
US7528223B2 (en) 2002-07-24 2009-05-05 Intercell Ag Antigens encoded by alternative reading frames from pathogenic viruses
EP1575504A4 (en) * 2002-08-01 2009-11-04 Us Gov Health & Human Serv Method of treating inflammatory arthropathies with suppressors of cpg oligonucleotides
AU2003250204B8 (en) 2002-08-02 2008-07-10 Glaxosmithkline Biologicals S.A. Neisserial vaccine compositions comprising a combination of antigens
CA2495570C (en) 2002-08-15 2012-12-04 3M Innovative Properties Company Immunostimulatory compositions and methods of stimulating an immune response
AR040996A1 (en) 2002-08-19 2005-04-27 Coley Pharm Group Inc IMMUNE STIMULATING NUCLEIC ACIDS
GB0220194D0 (en) 2002-08-30 2002-10-09 Chiron Spa Improved vesicles
JP2006504687A (en) 2002-09-13 2006-02-09 インターツェル・アクチェンゲゼルシャフト Method for isolating hepatitis C virus peptide
US20040106741A1 (en) * 2002-09-17 2004-06-03 Kriesel Joshua W. Nanofilm compositions with polymeric components
US8263091B2 (en) * 2002-09-18 2012-09-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Method of treating and preventing infections in immunocompromised subjects with immunostimulatory CpG oligonucleotides
AU2003268737A1 (en) * 2002-10-02 2004-04-23 Mochida Pharmaceutical Co., Ltd. Novel method of constructing monoclonal antibody
US8043622B2 (en) 2002-10-08 2011-10-25 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Method of treating inflammatory lung disease with suppressors of CpG oligonucleotides
EP2314604A3 (en) 2002-10-15 2011-05-25 Intercell AG Nucleic acids coding for adhesion factors of group B streptococcus, adhesion factors of group B streptococcus and further uses thereof
PL376792A1 (en) 2002-10-23 2006-01-09 Glaxosmithkline Biologicals S.A. Methods for vaccinating against malaria
US20060148064A1 (en) * 2002-10-25 2006-07-06 Srivastava Pramod K Apparatus and method for immunotherapy of a cancer through controlled cell lysis
DK2241325T3 (en) 2002-10-29 2012-04-10 Coley Pharm Group Inc Use of CPG oligonucleotides to treat Hepatitis Cvirus infection
US7758876B2 (en) * 2002-11-01 2010-07-20 The United States Of America As Represented By The Department Of Health And Human Services Method of preventing infections from bioterrorism agents with immunostimulatory CpG oligonucleotides
EP1562982B1 (en) 2002-11-15 2010-05-05 Novartis Vaccines and Diagnostics S.r.l. Unexpected surface proteins in neisseria meningitidis
NZ540276A (en) * 2002-11-21 2007-12-21 Bayhill Therapeutics Inc Methods and immune modulatory nucleic acid compositions for preventing and treating disease
GB0227346D0 (en) 2002-11-22 2002-12-31 Chiron Spa 741
US20040213808A1 (en) * 2002-12-11 2004-10-28 Michael Lieberman Recombinant vaccine against flavivirus infection
JP2006512927A (en) 2002-12-11 2006-04-20 コーリー ファーマシューティカル グループ，インコーポレイテッド 5 'CPG nucleic acids and methods of use thereof
US20050287170A1 (en) * 2002-12-11 2005-12-29 Hawaii Biotech, Inc. Subunit vaccine against West Nile viral infection
KR100525321B1 (en) * 2002-12-13 2005-11-02 안웅식 Pharmaceutical composition for prophylaxis and treatment of papillomavirus-derived diseases comprising papillomavirus antigen protein and CpG-oligodeoxynucleotide
EP1625140A4 (en) * 2002-12-23 2008-06-18 Dynavax Tech Corp Branched immunomodulatory compounds and methods of using the same
US8158768B2 (en) 2002-12-23 2012-04-17 Dynavax Technologies Corporation Immunostimulatory sequence oligonucleotides and methods of using the same
US7745606B2 (en) 2002-12-23 2010-06-29 Dynavax Technologies Corporation Immunostimulatory sequence oligonucleotides and methods of using the same
WO2004060396A2 (en) 2002-12-27 2004-07-22 Chiron Corporation Immunogenic compositions containing phospholpid
WO2004060319A2 (en) 2002-12-30 2004-07-22 3M Innovative Properties Company Immunostimulatory combinations
US7960522B2 (en) 2003-01-06 2011-06-14 Corixa Corporation Certain aminoalkyl glucosaminide phosphate compounds and their use
RU2544850C2 (en) 2003-01-06 2015-03-20 Корикса Корпорейшн Some amino alkyl glucose aminide phosphate derivatives and thereof application
EP2192172B1 (en) 2003-02-03 2014-12-03 iBio, Inc. System for expression of genes in plants
JP2006517974A (en) * 2003-02-13 2006-08-03 スリーエム イノベイティブ プロパティズ カンパニー Methods and compositions for IRM compounds and Toll-like receptor 8
EP1601756B1 (en) 2003-02-20 2010-12-15 University of Connecticut Health Center Methods for the preparation of alpha (2) macroglobulin-antigenic molecule complexes
GB2398783A (en) 2003-02-26 2004-09-01 Antonio Lanzavecchia A method for producing immortalised human B memory lymphocytes
US7485432B2 (en) * 2003-02-27 2009-02-03 3M Innovative Properties Company Selective modulation of TLR-mediated biological activity
CA2517518A1 (en) 2003-03-04 2004-09-16 Intercell Ag Streptococcus pyogenes antigens
US8110582B2 (en) 2003-03-04 2012-02-07 3M Innovative Properties Company Prophylactic treatment of UV-induced epidermal neoplasia
US8426457B2 (en) * 2003-03-13 2013-04-23 Medicis Pharmaceutical Corporation Methods of improving skin quality
AU2004220465A1 (en) 2003-03-13 2004-09-23 3M Innovative Properties Company Method of tattoo removal
US7704514B2 (en) 2003-03-24 2010-04-27 Intercell Ag Vaccines
US20040192585A1 (en) * 2003-03-25 2004-09-30 3M Innovative Properties Company Treatment for basal cell carcinoma
US7537767B2 (en) 2003-03-26 2009-05-26 Cytis Biotechnology Ag Melan-A- carrier conjugates
CN101675993A (en) * 2003-03-26 2010-03-24 赛托斯生物技术公司 melan-a peptide analogue-virus-like-particle conjugates
CA2520181A1 (en) * 2003-03-26 2004-10-14 Astral Inc. Selected rna motifs to include cell death and/or apoptosis
US7628994B2 (en) 2003-03-31 2009-12-08 Intercell Ag S. epidermidis antigens
CA2521050A1 (en) * 2003-04-02 2004-10-14 Coley Pharmaceutical Group, Ltd. Immunostimulatory nucleic acid oil-in-water formulations and related methods of use
US20040265351A1 (en) * 2003-04-10 2004-12-30 Miller Richard L. Methods and compositions for enhancing immune response
US7923560B2 (en) * 2003-04-10 2011-04-12 3M Innovative Properties Company Delivery of immune response modifier compounds
SE0301109D0 (en) * 2003-04-14 2003-04-14 Mallen Huang Nucleotide vaccine composition
CN1774447B (en) 2003-04-15 2011-04-06 英特塞尔股份公司 S. pneumoniae antigens
WO2004096144A2 (en) * 2003-04-28 2004-11-11 3M Innovative Properties Company Compositions and methods for induction of opioid receptors
EP1620460A2 (en) 2003-05-07 2006-02-01 Intercell AG Streptococcus agalactiae antigens i + ii
US7790189B2 (en) 2003-05-15 2010-09-07 Mitsui Sugar Co., Ltd. Immunostimulating agents
BRPI0410562B8 (en) 2003-05-22 2021-05-25 Fralunhofer Usa Inc carrier protein and its production method in a plant, isolated nucleic acid, expression vector and host cell
CA2525540A1 (en) 2003-05-30 2004-12-09 Intercell Ag Enterococcus antigens
JP5557415B2 (en) 2003-06-02 2014-07-23 ノバルティス バクシンズ アンド ダイアグノスティックス，インコーポレーテッド Immunogenic compositions based on microparticles containing adsorbed toxoid and polysaccharide-containing antigens
WO2005000884A1 (en) 2003-06-05 2005-01-06 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Poly-gamma-glutamic conjugates for eliciting immune responses directed against bacilli
EP1635846A4 (en) * 2003-06-20 2009-01-28 Coley Pharm Gmbh Small molecule toll-like receptor (tlr) antagonists
DE602004031017D1 (en) * 2003-07-10 2011-02-24 Cytos Biotechnology Ag COMPOSITION PACKAGED VIRUSIC PARTICLES INCLUDING THE IMPROVEMENT OF AN IMMUNE RESPONSE
US20050013812A1 (en) * 2003-07-14 2005-01-20 Dow Steven W. Vaccines using pattern recognition receptor-ligand:lipid complexes
CA2534313C (en) * 2003-08-05 2013-03-19 3M Innovative Properties Company Formulations containing an immune response modifier
EP1653914A4 (en) * 2003-08-12 2008-10-29 3M Innovative Properties Co Oxime substituted imidazo-containing compounds
US20060035242A1 (en) 2004-08-13 2006-02-16 Michelitsch Melissa D Prion-specific peptide reagents
US20050048072A1 (en) * 2003-08-25 2005-03-03 3M Innovative Properties Company Immunostimulatory combinations and treatments
ES2406730T3 (en) * 2003-08-27 2013-06-07 3M Innovative Properties Company Imidazoquinolines substituted with aryloxy and arylalkylenoxy
US20060216333A1 (en) * 2003-09-02 2006-09-28 Miller Richard L Methods related to the treatment of mucosal associated conditions
JP2007504269A (en) * 2003-09-05 2007-03-01 スリーエム イノベイティブ プロパティズ カンパニー Method for treating CD5 + B cell lymphoma
AU2004274430A1 (en) 2003-09-12 2005-03-31 Antigenics, Inc. Vaccine for treatment and prevention of herpes simplex virus infection
US7615539B2 (en) * 2003-09-25 2009-11-10 Coley Pharmaceutical Group, Inc. Nucleic acid-lipophilic conjugates
CA2539715C (en) 2003-10-02 2015-02-24 Glaxosmithkline Biologicals S.A. Pertussis antigens and use thereof in vaccination
US7544697B2 (en) * 2003-10-03 2009-06-09 Coley Pharmaceutical Group, Inc. Pyrazolopyridines and analogs thereof
US20090075980A1 (en) * 2003-10-03 2009-03-19 Coley Pharmaceutical Group, Inc. Pyrazolopyridines and Analogs Thereof
US8871782B2 (en) * 2003-10-03 2014-10-28 3M Innovative Properties Company Alkoxy substituted imidazoquinolines
EP1675614A2 (en) * 2003-10-11 2006-07-05 Inex Pharmaceuticals Corp. Methods and compositions for enhancing innate immunity and antibody dependent cellular cytotoxicity
SG122973A1 (en) * 2003-10-30 2006-06-29 Coley Pharm Gmbh C-class oligonucleotide analogs with enhanced immunostimulatory potency
AU2004285575A1 (en) * 2003-10-31 2005-05-12 3M Innovative Properties Company Neutrophil activation by immune response modifier compounds
US20050239733A1 (en) * 2003-10-31 2005-10-27 Coley Pharmaceutical Gmbh Sequence requirements for inhibitory oligonucleotides
US20050100983A1 (en) * 2003-11-06 2005-05-12 Coley Pharmaceutical Gmbh Cell-free methods for identifying compounds that affect toll-like receptor 9 (TLR9) signaling
EP1685129A4 (en) 2003-11-14 2008-10-22 3M Innovative Properties Co Oxime substituted imidazo ring compounds
CA2545825A1 (en) * 2003-11-14 2005-06-02 3M Innovative Properties Company Hydroxylamine substituted imidazo ring compounds
WO2005051317A2 (en) * 2003-11-25 2005-06-09 3M Innovative Properties Company Substituted imidazo ring systems and methods
US20050287118A1 (en) * 2003-11-26 2005-12-29 Epitomics, Inc. Bacterial plasmid with immunological adjuvant function and uses thereof
US20050277127A1 (en) * 2003-11-26 2005-12-15 Epitomics, Inc. High-throughput method of DNA immunogen preparation and immunization
WO2005055932A2 (en) * 2003-12-02 2005-06-23 3M Innovative Properties Company Therapeutic combinations and methods including irm compounds
US20050226878A1 (en) * 2003-12-02 2005-10-13 3M Innovative Properties Company Therapeutic combinations and methods including IRM compounds
ATE554106T1 (en) 2003-12-23 2012-05-15 Arbor Vita Corp ANTIBODIES FOR ONCOGENIC STRAINS OF HPV AND METHOD OF USE THEREOF
JP4817599B2 (en) * 2003-12-25 2011-11-16 独立行政法人科学技術振興機構 Immune activity enhancer and method for enhancing immune activity using the same
WO2005066170A1 (en) * 2003-12-29 2005-07-21 3M Innovative Properties Company Arylalkenyl and arylalkynyl substituted imidazoquinolines
US8735421B2 (en) * 2003-12-30 2014-05-27 3M Innovative Properties Company Imidazoquinolinyl sulfonamides
JP2007517055A (en) * 2003-12-30 2007-06-28 スリーエム イノベイティブ プロパティズ カンパニー Enhanced immune response
WO2005072290A2 (en) * 2004-01-23 2005-08-11 Joslin Diabetes Center Methods of treating, reducing, or preventing autoimmune conditions
CA2555274A1 (en) 2004-02-05 2005-08-25 The Ohio State University Research Foundation Chimeric vegf peptides
US20050181035A1 (en) * 2004-02-17 2005-08-18 Dow Steven W. Systemic immune activation method using non CpG nucleic acids
TW200533750A (en) * 2004-02-19 2005-10-16 Coley Pharm Group Inc Immunostimulatory viral RNA oligonucleotides
WO2005090968A1 (en) * 2004-03-16 2005-09-29 Inist Inc. Tat-based immunomodulatory compositions and methods of their discovery and use
WO2005092360A1 (en) 2004-03-19 2005-10-06 Xiu-Min Li Herbal therapy for the treatment of food allergy
US8697873B2 (en) * 2004-03-24 2014-04-15 3M Innovative Properties Company Amide substituted imidazopyridines, imidazoquinolines, and imidazonaphthyridines
TWI235440B (en) * 2004-03-31 2005-07-01 Advanced Semiconductor Eng Method for making leadless semiconductor package
JP2007532572A (en) * 2004-04-09 2007-11-15 スリーエム イノベイティブ プロパティズ カンパニー Methods, compositions and preparations for delivering immune response modifiers
WO2006126981A2 (en) * 2004-04-28 2006-11-30 3M Innovative Properties Company Compositions and methods for mucosal vaccination
JP5014122B2 (en) 2004-05-06 2012-08-29 アメリカ合衆国 Methods and compositions for the treatment of uveitis
WO2006031264A2 (en) 2004-05-25 2006-03-23 Oregon Health And Science University Siv and hiv vaccination using rhcmv- and hcmv-based vaccine vectors
US20050267145A1 (en) * 2004-05-28 2005-12-01 Merrill Bryon A Treatment for lung cancer
US20080154210A1 (en) 2004-05-28 2008-06-26 Oryxe Mixture for Transdermal Delivery of Low and High Molecular Weight Compounds
US8017779B2 (en) * 2004-06-15 2011-09-13 3M Innovative Properties Company Nitrogen containing heterocyclyl substituted imidazoquinolines and imidazonaphthyridines
WO2006009826A1 (en) * 2004-06-18 2006-01-26 3M Innovative Properties Company Aryloxy and arylalkyleneoxy substituted thiazoloquinolines and thiazolonaphthyridines
WO2006009832A1 (en) * 2004-06-18 2006-01-26 3M Innovative Properties Company Substituted imidazo ring systems and methods
US7897609B2 (en) * 2004-06-18 2011-03-01 3M Innovative Properties Company Aryl substituted imidazonaphthyridines
WO2006065280A2 (en) * 2004-06-18 2006-06-22 3M Innovative Properties Company Isoxazole, dihydroisoxazole, and oxadiazole substituted imidazo ring compounds and methods
NZ553244A (en) * 2004-07-18 2009-10-30 Csl Ltd Immuno stimulating complex and oligonucleotide formulations for inducing enhanced interferon-gamma responses
DK2412728T3 (en) 2004-08-03 2015-04-07 Innate Pharma Therapeutic compositions for cancer, that target 4Ig-B7-H3
GB0417494D0 (en) 2004-08-05 2004-09-08 Glaxosmithkline Biolog Sa Vaccine
US20060045886A1 (en) * 2004-08-27 2006-03-02 Kedl Ross M HIV immunostimulatory compositions
US20090270443A1 (en) * 2004-09-02 2009-10-29 Doris Stoermer 1-amino imidazo-containing compounds and methods
CA2580103C (en) 2004-09-22 2021-11-16 Glaxosmithkline Biologicals S.A. Immunogenic composition
ATE465173T1 (en) 2004-09-24 2010-05-15 Intercell Ag ALTERED VP-1CAPSID PROTEIN FROM PARVOVIRUS B19
JP2008000001A (en) * 2004-09-30 2008-01-10 Osaka Univ Immune stimulating oligonucleotide and use in pharmaceutical
WO2006042254A2 (en) * 2004-10-08 2006-04-20 3M Innovative Properties Company Adjuvant for dna vaccines
EP2808384B1 (en) 2004-10-08 2017-12-06 The Government of the United States of America as represented by the Secretary of the Department of Health and Human Services Modulation of replicative fitness by using less frequently used synonymous codons
MY159370A (en) * 2004-10-20 2016-12-30 Coley Pharm Group Inc Semi-soft-class immunostimulatory oligonucleotides
US7332324B2 (en) 2004-12-03 2008-02-19 University Of Toledo Attenuated vaccine useful for immunizations against Coccidioides spp. infections
US20110070575A1 (en) * 2004-12-08 2011-03-24 Coley Pharmaceutical Group, Inc. Immunomodulatory Compositions, Combinations and Methods
WO2006065751A2 (en) 2004-12-13 2006-06-22 Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Cpg oligonucleotide prodrugs, compositions thereof and associated therapeutic methods
US7943609B2 (en) 2004-12-30 2011-05-17 3M Innovative Proprerties Company Chiral fused [1,2]imidazo[4,5-C] ring compounds
AU2005326708C1 (en) 2004-12-30 2012-08-30 3M Innovative Properties Company Substituted chiral fused [1,2]imidazo[4,5-c] ring compounds
ES2538498T3 (en) 2004-12-30 2015-06-22 Meda Ab Use of Imiquimod for the treatment of skin metastases from a breast cancer tumor
NZ555937A (en) 2005-01-27 2009-05-31 Childrens Hosp & Res Ct Oak GNA1870-based vesicle vaccines for broad spectrum protection against diseases caused by Neisseria meningitidis
AU2006210392A1 (en) * 2005-02-04 2006-08-10 Coley Pharmaceutical Group, Inc. Aqueous gel formulations containing immune response modifiers
US7968563B2 (en) 2005-02-11 2011-06-28 3M Innovative Properties Company Oxime and hydroxylamine substituted imidazo[4,5-c] ring compounds and methods
JP2008531018A (en) * 2005-02-24 2008-08-14 コーリー ファーマシューティカル グループ，インコーポレイテッド Immunostimulatory oligonucleotide
GB0504436D0 (en) 2005-03-03 2005-04-06 Glaxosmithkline Biolog Sa Vaccine
US8101345B1 (en) 2005-03-25 2012-01-24 Isis Pharmaceuticals, Inc. Proinflammatory nucleic acids
CA2602637A1 (en) 2005-03-31 2006-10-05 Glaxosmithkline Biologicals Sa Vaccines against chlamydial infection
US7943610B2 (en) 2005-04-01 2011-05-17 3M Innovative Properties Company Pyrazolopyridine-1,4-diamines and analogs thereof
JP2008538550A (en) 2005-04-01 2008-10-30 コーリー ファーマシューティカル グループ，インコーポレイテッド 1-Substituted pyrazolo (3,4-c) cyclic compounds as modulators of cytokine biosynthesis for treating viral infections and neoplastic diseases
US8642577B2 (en) * 2005-04-08 2014-02-04 Chimerix, Inc. Compounds, compositions and methods for the treatment of poxvirus infections
KR20080008350A (en) * 2005-04-08 2008-01-23 콜레이 파마시티컬 그룹, 인코포레이티드 Methods for treating infectious disease exacerbated asthma
EP2842559A3 (en) * 2005-04-08 2015-03-18 Chimerix, Inc. Compounds, compositions and methods for the treatment of viral infections and other medical disorders
AU2006241166A1 (en) * 2005-04-25 2006-11-02 3M Innovative Properties Company Immunostimulatory compositions
AU2006241149A1 (en) * 2005-04-26 2006-11-02 Coley Pharmaceutical Gmbh Modified oligoribonucleotide analogs with enhanced immunostimulatory activity
NZ562729A (en) 2005-04-29 2009-10-30 Infectious Disease Res Inst Id Novel method for preventing or treating M tuberculosis infection using Mtb72f fusion proteins
CN101238145B (en) 2005-06-14 2013-04-24 普罗陶克斯有限公司 Use of modified pore-forming proteins in preparing medicine for treating or preventing benign prostatic hyperplasia
AU2006261342B2 (en) 2005-06-15 2012-02-02 The Ohio State University Research Foundation Her-2 peptides
GB0513421D0 (en) 2005-06-30 2005-08-03 Glaxosmithkline Biolog Sa Vaccines
ATE476193T1 (en) 2005-07-01 2010-08-15 Index Pharmaceuticals Ab IMMUNO-STIMULATORY PROCEDURE
ES2435531T3 (en) 2005-07-01 2013-12-20 Index Pharmaceuticals Ab Modulation of steroid responsiveness
WO2007008463A2 (en) * 2005-07-07 2007-01-18 Coley Pharmaceutical Group, Inc. Anti-ctla-4 antibody and cpg-motif-containing synthetic oligodeoxynucleotide combination therapy for cancer treatment
US20080206276A1 (en) 2005-07-08 2008-08-28 Michael Otto Targeting Poly-Gamma-Glutamic Acid to Treat Staphylococcus Epidermidis and Related Infections
AU2006268333B2 (en) 2005-07-11 2012-06-14 Globeimmune, Inc. Compositions and methods for eliciting an immune response to escape mutants of targeted therapies
CN101378781B (en) 2005-08-03 2013-08-07 美国弗劳恩霍夫股份有限公司 Compositions and methods for production of immunoglobulins
US20100130425A1 (en) 2005-09-09 2010-05-27 Oregon Health & Science University Use of toll-like receptor ligands in treating excitotoxic injury, ischemia and/or hypoxia
EA013468B1 (en) * 2005-09-16 2010-04-30 Коли Фармасьютикал Гмбх Immunostimulatory single-stranded ribonucleic acid with phosphodiester backbone
KR20080047463A (en) * 2005-09-16 2008-05-28 콜리 파마슈티칼 게엠베하 Modulation of immunostimulatory properties of short interfering ribonucleic acid (sirna) by nucleotide modification
US20070081972A1 (en) * 2005-09-30 2007-04-12 The University Of Iowa Research Foundation Polymer-based delivery system for immunotherapy of cancer
WO2007050034A1 (en) 2005-10-28 2007-05-03 Index Pharmaceuticals Ab Composition and method for the prevention, treatment and/or alleviation of an inflammatory disease
PT1957647E (en) 2005-11-25 2015-06-01 Zoetis Belgium S A Immunostimulatory oligoribonucleotides
HUE051267T2 (en) 2005-12-13 2021-03-01 Harvard College Scaffolds for cell transplantation
NZ569741A (en) 2005-12-14 2012-02-24 Cytos Biotechnology Ag Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity
LT2384765T (en) 2005-12-22 2017-01-10 Glaxosmithkline Biologicals S.A. Streptococcus pneumoniae vaccine
GB0607088D0 (en) 2006-04-07 2006-05-17 Glaxosmithkline Biolog Sa Vaccine
WO2007082105A2 (en) 2006-01-16 2007-07-19 Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Chlamydia vaccine
SG175492A1 (en) 2006-01-17 2011-11-28 Arne Forsgren A novel surface exposed haemophilus influenzae protein (protein e; pe)
CA2642054C (en) 2006-02-13 2017-11-21 Fraunhofer Usa, Inc. Influenza antigens, vaccine compositions, and related methods
US20080045473A1 (en) 2006-02-15 2008-02-21 Coley Pharmaceutical Gmbh Compositions and methods for oligonucleotide formulations
WO2007100634A2 (en) * 2006-02-22 2007-09-07 3M Innovative Properties Company Immune response modifier conjugates
US7842299B2 (en) 2006-03-14 2010-11-30 Oregon Health & Science University Methods for producing an immune response to tuberculosis
JP2009531387A (en) 2006-03-30 2009-09-03 グラクソスミスクライン バイオロジカルズ ソシエテ アノニム Immunogenic composition
US20100016250A1 (en) 2006-04-14 2010-01-21 Kyowa Hakko Kirin Co., Ltd. Toll-like receptor 9 agonists
CA2653939C (en) 2006-05-31 2013-01-22 Toray Industries, Inc. Immunostimulatory oligonucleotides and use thereof in pharmaceuticals
MX2008015372A (en) 2006-06-02 2009-03-23 Glaxosmithkline Biolog Sa Method for identifying whether a patient will be responder or not to immunotherapy.
KR100906970B1 (en) 2006-06-03 2009-07-10 주식회사 바이오씨에스 Therapeutic use of CpG oligodeoxynucleotide for skin immune disease
WO2007143582A2 (en) * 2006-06-05 2007-12-13 Baylor College Of Medicine Reversal of the suppressive function of specific t cells via toll-like receptor 8 signaling
ES2427994T3 (en) 2006-06-12 2013-11-05 Cytos Biotechnology Ag Processes for packaging oligonucleotides into viral bacterial phage particles
WO2007144316A2 (en) 2006-06-12 2007-12-21 Glaxosmithkline Biologicals Sa Vaccine
CN101490083A (en) 2006-07-07 2009-07-22 英特塞尔股份公司 Small streptococcus pyogenes antigens and their use
WO2008094183A2 (en) * 2006-07-11 2008-08-07 University Of Connecticut Use of conditional plasmodium strains lacking nutrient transporters in malaria vaccination
US8153116B2 (en) 2006-07-11 2012-04-10 University Of Connecticut Use of conditional plasmodium strains lacking an essential gene in malaria vaccination
WO2008008432A2 (en) * 2006-07-12 2008-01-17 Coley Pharmaceutical Group, Inc. Substituted chiral fused( 1,2) imidazo (4,5-c) ring compounds and methods
DE102006035618A1 (en) * 2006-07-31 2008-02-07 Curevac Gmbh New nucleic acid useful as immuno-stimulating adjuvant for manufacture of a composition for treatment of cancer diseases e.g. colon carcinomas and infectious diseases e.g. influenza and malaria
WO2008014979A2 (en) 2006-07-31 2008-02-07 Curevac Gmbh NUCLEIC ACID OF FORMULA (I): GIXmGn, OR (II): CIXmCn, IN PARTICULAR AS AN IMMUNE-STIMULATING AGENT/ADJUVANT
AU2007293672B2 (en) 2006-09-07 2013-06-27 Glaxosmithkline Biologicals S.A. Vaccine
JP2010503384A (en) 2006-09-15 2010-02-04 インターセル アーゲー Borrelia antigen
US20090181078A1 (en) 2006-09-26 2009-07-16 Infectious Disease Research Institute Vaccine composition containing synthetic adjuvant
PL2468300T3 (en) 2006-09-26 2018-04-30 Infectious Disease Research Institute Vaccine composition containing synthetic adjuvant
MX2009003398A (en) * 2006-09-27 2009-08-12 Coley Pharm Gmbh Cpg oligonucleotide analogs containing hydrophobic t analogs with enhanced immunostimulatory activity.
US20100144846A1 (en) * 2006-10-26 2010-06-10 Coley Pharmaceutical Gmbh Oligoribonucleotides and uses thereof
ES2761949T3 (en) 2006-11-01 2020-05-21 Ventana Med Syst Inc Haptens, hapten conjugates, compositions thereof and method for their preparation and use
US20090142362A1 (en) * 2006-11-06 2009-06-04 Avant Immunotherapeutics, Inc. Peptide-based vaccine compositions to endogenous cholesteryl ester transfer protein (CETP)
EP2094849B1 (en) 2006-11-09 2014-01-08 Dynavax Technologies Corporation Long term disease modification using immunostimulatory oligonucleotides
EP1923069A1 (en) 2006-11-20 2008-05-21 Intercell AG Peptides protective against S. pneumoniae and compositions, methods and uses relating thereto
US20080149123A1 (en) 2006-12-22 2008-06-26 Mckay William D Particulate material dispensing hairbrush with combination bristles
EP2266591A3 (en) 2007-01-12 2012-08-08 Intercell AG Protective proteins of S. agalactiae, combinations thereof and methods of using the same
EP2125868B1 (en) 2007-02-28 2015-06-10 The Govt. Of U.S.A. As Represented By The Secretary Of The Department Of Health And Human Services Brachyury polypeptides and methods for use
CA2679410C (en) 2007-03-02 2017-01-17 Glaxosmithkline Biologicals S.A. Novel method and compositions
KR20100051041A (en) * 2007-03-07 2010-05-14 엔벤타 바이오파마슈티칼스 코퍼레이션 Double-stranded locked nucleic acid compositions
RU2505313C2 (en) 2007-03-19 2014-01-27 Глоубиммьюн, Инк. Compositions and methods for target elimination of mutation escape in target cancer therapy
JP5378350B2 (en) 2007-04-04 2013-12-25 インフェクティアス ディジーズ リサーチ インスティチュート Immunogenic composition comprising Mycobacterium tuberculosis polypeptide and fusions thereof
US9452209B2 (en) 2007-04-20 2016-09-27 Glaxosmithkline Biologicals Sa Influenza vaccine
BRPI0810865A2 (en) 2007-04-28 2017-05-09 Fraunhofer Usa Inc trypanosome antigens, vaccine compositions, and related methods
US8236326B2 (en) 2007-05-02 2012-08-07 Intercell Ag Klebsiella antigens
AR066376A1 (en) 2007-05-02 2009-08-12 Glaxosmithkline Biolog Sa VACCINE
EP2573176B1 (en) * 2007-05-04 2016-04-06 InDex Pharmaceuticals AB Tumour growth inhibitory compounds and methods of their use
BRPI0811621A2 (en) 2007-05-17 2014-10-21 Coley Pharm Group Inc CLASS A OLIGONUCLEOTIDES WITH IMMUNOSTIMULATING POWER, COMPOSITION CONTAINING THEM AND USE OF SUCH COMPOSITION
ES2544484T3 (en) * 2007-05-18 2015-08-31 Adiutide Pharmaceuticals Gmbh Phosphate modified oligonucleotide analogues with immunostimulatory activity
EP3561513A1 (en) 2007-05-23 2019-10-30 Ventana Medical Systems, Inc. Polymeric carriers for immunohistochemistry and in situ hybridization
UY31101A1 (en) 2007-05-24 2009-01-05 Glaxosmithkline Biologicals Sa ANTIGENIC LIOFILIZED COMPOSITION
EP2012122A1 (en) 2007-07-06 2009-01-07 Medigene AG Mutated parvovirus structural proteins as vaccines
WO2008145400A2 (en) 2007-05-31 2008-12-04 Medigene Ag Mutated structural protein of a parvovirus
CA2690271A1 (en) 2007-06-18 2008-12-24 Intercell Ag Chlamydia antigens
EA020817B1 (en) 2007-06-26 2015-02-27 Глаксосмитклайн Байолоджикалс С.А. Vaccine, comprising streptococcus pneumoniae capsular polysaccharide conjugates
ES2725450T3 (en) 2007-07-02 2019-09-24 Etubics Corp Methods and compositions for the production of an adenoviral vector for use in multiple vaccinations
EP2178558B1 (en) 2007-07-11 2014-04-30 iBio, Inc. Yersinia pestis antigens, vaccine compositions, and related methods
MX2010001284A (en) 2007-08-02 2010-08-31 Biondvax Pharmaceuticals Ltd Multimeric multiepitope influenza vaccines.
JP2010535504A (en) 2007-08-03 2010-11-25 プレジデント アンド フェロウズ オブ ハーバード カレッジ Chlamydia antigen
US7879812B2 (en) 2007-08-06 2011-02-01 University Of Iowa Research Foundation Immunomodulatory oligonucleotides and methods of use therefor
CA2695477A1 (en) 2007-08-13 2009-02-19 Glaxosmithkline Biologicals S.A. Infant plasmodium falciparum cs vaccines
WO2009026465A2 (en) * 2007-08-21 2009-02-26 Dynavax Technologies Corporation Composition and methods of making and using influenza proteins
EP2185160B1 (en) * 2007-08-31 2019-02-13 Neurimmune Holding AG Method of providing patient specific immune response in amyloidoses and protein aggregation disorders
WO2009030254A1 (en) 2007-09-04 2009-03-12 Curevac Gmbh Complexes of rna and cationic peptides for transfection and for immunostimulation
BRPI0817008A2 (en) 2007-09-17 2017-05-16 Glaxosmithkline Biologicals Sa oligonucleotide, primer or probe, primer pair, kit, methods for detecting the presence and / or amount of the unmethylated and methylated mage-a3 gene in a DNA-containing sample to diagnose cancer or cancer predisposition or cancer predisposition, to identify and / or select a suitable patient for treatment with a mage-a3 immunotherapeutic, to predict the likelihood of successful cancer treatment, to select an appropriate cancer treatment regimen, to treat cancer in a patient, to treat a patient susceptible to recurrence of a tumor expressing mage-a3, and use of a composition
KR20100068422A (en) * 2007-10-09 2010-06-23 콜리 파마슈티칼 게엠베하 Immune stimulatory oligonucleotide analogs containing modified sugar moieties
EA026990B1 (en) 2007-11-07 2017-06-30 Селлдекс Терапьютикс Инк. Antibodies which bind to human cell dec-205
CA2703621C (en) 2007-11-09 2022-03-22 The Salk Institute For Biological Studies Use of tam receptor inhibitors as antimicrobials
EP2227250A4 (en) 2007-12-03 2011-07-06 Harvard College Chlamydia antigens
ES2664753T3 (en) 2007-12-07 2018-04-23 Glaxosmithkline Biologicals Sa Immune response induction compositions
MX2010007107A (en) 2007-12-24 2010-12-21 Id Biomedical Corp Quebec Recombinant rsv antigens.
WO2009094191A2 (en) * 2008-01-25 2009-07-30 Chimerix, Inc. Methods of treating viral infections
RU2545701C2 (en) 2008-01-31 2015-04-10 Куревак Гмбх NUCLEIC ACIDS OF FORMULA (I) (NuGlXmGnNv)a AND DERIVATIVES THEREOF AS IMMUNOSTIMULATING AGENTS/ADJUVANTS
WO2009102465A2 (en) 2008-02-13 2009-08-20 President And Fellows Of Harvard College Continuous cell programming devices
US10383887B2 (en) 2008-02-20 2019-08-20 New York University Preventing and treating amyloid-beta deposition by stimulation of innate immunity
CA2717870A1 (en) 2008-03-10 2009-09-17 Children's Hospital & Research Center At Oakland Chimeric factor h binding proteins (fhbp) containing a heterologous b domain and methods of use
BRPI0909664A2 (en) 2008-03-17 2019-09-24 Intercell Ag protective peptides against s. pneumoniae and related compositions, methods and uses thereof
CN102065880B (en) 2008-04-18 2015-11-25 综合医院公司 Use the immunization therapy of oneself's assembling vaccine
WO2009140626A2 (en) 2008-05-15 2009-11-19 Dynavax Technologies Corporation Long term disease modification using immunostimulatory oligonucleotides
US8222225B2 (en) 2008-05-21 2012-07-17 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Method of treating pneumoconiosis with oligodeoxynucleotides
EP2293813A4 (en) 2008-05-23 2012-07-11 Univ Michigan Nanoemulsion vaccines
CA2720728C (en) 2008-06-05 2018-04-03 Ventana Medical Systems, Inc. Compositions comprising nanomaterials and method for using such compositions for histochemical processes
TWI351288B (en) * 2008-07-04 2011-11-01 Univ Nat Pingtung Sci & Tech Cpg dna adjuvant in avian vaccines
KR101707569B1 (en) 2008-08-01 2017-02-16 감마 백신즈 피티와이 리미티드 Influenza vaccines
EP2353008A2 (en) 2008-09-22 2011-08-10 Oregon Health and Science University Methods for detecting a mycobacterium tuberculosis infection
JP5722782B2 (en) 2008-09-26 2015-05-27 ナノバイオ コーポレーション Nanoemulsion therapeutic composition and method of use thereof
WO2010037046A1 (en) 2008-09-28 2010-04-01 Fraunhofer Usa, Inc. Humanized neuraminidase antibody and methods of use thereof
WO2010037408A1 (en) 2008-09-30 2010-04-08 Curevac Gmbh Composition comprising a complexed (m)rna and a naked mrna for providing or enhancing an immunostimulatory response in a mammal and uses thereof
NZ591925A (en) * 2008-10-16 2012-09-28 Internat Vaccine Inst Adjuvant composition comprising a host defense peptide, an immunostimulatory sequence and a polyanionic polymer
US9732324B2 (en) 2008-10-23 2017-08-15 Cornell University Anti-viral method
WO2010048731A1 (en) * 2008-10-31 2010-05-06 Christopher John Ong Aminoacyl trna-synthetase inhibitors as broad-spectrum immunosuppressive agents
EP2376089B1 (en) 2008-11-17 2018-03-14 The Regents of the University of Michigan Cancer vaccine compositions and methods of using the same
EP3936122A1 (en) 2008-11-24 2022-01-12 Massachusetts Institute Of Technology Methods and compositions for localized agent delivery
ES2616051T3 (en) 2008-12-02 2017-06-09 Wave Life Sciences Japan, Inc. Method for the synthesis of modified nucleic acids in the phosphorus atom
CN102239253A (en) 2008-12-03 2011-11-09 普罗蒂亚维仕尼科技有限公司 Glutamyl trna synthetase (GTS) fragments
CN102325790A (en) 2008-12-03 2012-01-18 西马生物医学计划公司 Use of phenol-soluble modulins for vaccine development
US8552165B2 (en) * 2008-12-09 2013-10-08 Heather Davis Immunostimulatory oligonucleotides
MY172421A (en) 2008-12-09 2019-11-25 Coley Pharm Group Inc Immunostimulatory oligonucleotides
PE20142330A1 (en) * 2008-12-09 2015-01-14 Pfizer Vaccines Llc IGE CH3 PEPTIDE VACCINE
BRPI1005670A8 (en) 2009-01-05 2017-12-26 Epitogenesis Inc adjuvant compositions and processes of use.
BRPI1006901A2 (en) 2009-01-20 2016-02-16 Transgène S A ex vivo method and kit for testing if a patient will respond therapeutically to a treatment method, and use of sicam-1 levels in a blood sample
US20100234283A1 (en) 2009-02-04 2010-09-16 The Ohio State University Research Foundation Immunogenic epitopes, peptidomimetics, and anti-peptide antibodies, and methods of their use
US20130243779A1 (en) 2009-02-05 2013-09-19 Intercell Ag Peptides protective against e. faecalis, methods and uses relating thereto
WO2010092176A2 (en) 2009-02-13 2010-08-19 Intercell Ag Nontypable haemophilus influenzae antigens
US8664183B2 (en) 2009-02-27 2014-03-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services SPANX-B polypeptides and their use
WO2010105815A2 (en) 2009-03-17 2010-09-23 Oncomethylome Sciences S.A. Improved detection of gene expression
DK2411043T3 (en) 2009-03-23 2013-10-21 Pin Pharma Inc TREATMENT OF CANCER WITH IMMUNOSTIMULATORY HIV TAT DERIVATE POLYPEPTIDES
JP5774578B2 (en) 2009-03-24 2015-09-09 トランジェーヌ、ソシエテ、アノニムＴｒａｎｓｇｅｎｅ Ｓ．Ａ． Biomarkers for monitoring patients
AU2010232490B2 (en) 2009-04-03 2016-08-11 Agenus Inc. Methods for preparing and using multichaperone-antigen complexes
JP5661738B2 (en) 2009-04-17 2015-01-28 トランジェーヌ、ソシエテ、アノニムＴｒａｎｓｇｅｎｅ Ｓ．Ａ． Biomarkers for monitoring patients
AU2010239689A1 (en) * 2009-04-21 2011-11-03 Selecta Biosciences, Inc. Immunonanotherapeutics providing a Th1-biased response
EP2424562B1 (en) 2009-04-30 2015-10-07 Coley Pharmaceutical Group, Inc. Pneumococcal vaccine and uses thereof
EA022699B1 (en) 2009-05-27 2016-02-29 Селекта Байосайенсиз, Инк. Targeted synthetic nanocarriers with ph sensitive release of immunomodulatory agents
EP2435469A1 (en) 2009-05-27 2012-04-04 GlaxoSmithKline Biologicals S.A. Casb7439 constructs
US8722064B2 (en) 2009-06-05 2014-05-13 Infectious Disease Research Institute Synthetic glucopyranosyl lipid adjuvants
US20100316673A1 (en) 2009-06-16 2010-12-16 The Regents Of The University Of Michigan Nanoemulsion vaccines
EA023054B1 (en) 2009-06-24 2016-04-29 Глэксосмитклайн Байолоджикалз С.А. Recombinant rsv antigens
US8889146B2 (en) 2009-06-24 2014-11-18 Glaxosmithkline Biologicals, Sa Vaccine
US9744183B2 (en) 2009-07-06 2017-08-29 Wave Life Sciences Ltd. Nucleic acid prodrugs and methods of use thereof
SG176619A1 (en) 2009-07-10 2012-01-30 Transgene Sa Biomarker for selecting patients and related methods
TW201116294A (en) 2009-07-15 2011-05-16 Novartis Ag RSV F protein compositions and methods for making same
US8614200B2 (en) 2009-07-21 2013-12-24 Chimerix, Inc. Compounds, compositions and methods for treating ocular conditions
CA2768346A1 (en) 2009-07-30 2011-02-03 Pfizer Vaccines Llc Antigenic tau peptides and uses thereof
US20110033515A1 (en) * 2009-08-04 2011-02-10 Rst Implanted Cell Technology Tissue contacting material
GB0913681D0 (en) 2009-08-05 2009-09-16 Glaxosmithkline Biolog Sa Immunogenic composition
GB0913680D0 (en) 2009-08-05 2009-09-16 Glaxosmithkline Biolog Sa Immunogenic composition
EP2471801A4 (en) 2009-08-26 2013-12-04 Rna Inc Lipoteichoic acid-derived glycolipids, and compositions comprising same
WO2011026111A1 (en) 2009-08-31 2011-03-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Oral delivery of a vaccine to the large intestine to induce mucosal immunity
US20110053829A1 (en) 2009-09-03 2011-03-03 Curevac Gmbh Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
DK2473605T3 (en) 2009-09-03 2018-05-28 Pfizer Vaccines Llc PCSK9-VACCINE
EP2477642A4 (en) * 2009-09-17 2013-03-13 Mutual Pharmaceutical Co Method of treating asthma with antiviral agents
GB0917457D0 (en) 2009-10-06 2009-11-18 Glaxosmithkline Biolog Sa Method
CA2776144C (en) 2009-09-29 2020-10-27 Fraunhofer Usa, Inc. Influenza hemagglutinin antibodies, compositions, and related methods
CA2776922A1 (en) 2009-10-07 2011-04-14 Uvic Industry Partnerships Inc. Vaccines comprising heat-sensitive transgenes
MX2012004090A (en) 2009-10-09 2012-04-20 Sanofi Sa Polypeptides for binding to the "receptor for advanced glycation endproducts" as well as compositions and methods involving the same.
EP2308896A1 (en) 2009-10-09 2011-04-13 Sanofi-aventis Polypeptides for binding to the "receptor for advanced glycation endproducts" as well as compositions and methods involving the same
EP2319871A1 (en) 2009-11-05 2011-05-11 Sanofi-aventis Polypeptides for binding to the "receptor for advanced glycation endproducts" as well as compositions and methods involving the same
US9181306B2 (en) 2009-10-16 2015-11-10 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Insertion of foreign genes in rubella virus and their stable expression in a live, attenuated viral vaccine
GB0919117D0 (en) 2009-10-30 2009-12-16 Glaxosmithkline Biolog Sa Process
EP2502076B1 (en) 2009-11-20 2018-04-18 Oregon Health and Science University Methods for detecting a mycobacterium tuberculosis infection
WO2011067758A2 (en) 2009-12-02 2011-06-09 Protea Vaccine Technologies Ltd. Immunogenic fragments and multimers from streptococcus pneumoniae proteins
NZ629256A (en) 2009-12-22 2016-02-26 Celldex Therapeutics Inc Vaccine compositions
FR2954703B1 (en) 2009-12-28 2013-12-13 Chu Nantes TLR 4 AND 9 RECEPTOR AGONISTS TO PREVENT SEPTIC COMPLICATIONS OF POSTTRAUMATIC IMMUNODEPRESSION IN HOSPITAL PATIENTS FOR SEVERE INJURIES
ES2629165T3 (en) 2010-02-12 2017-08-07 Chimerix, Inc. Methods of treating a viral infection
WO2011101332A1 (en) 2010-02-16 2011-08-25 Proyecto De Biomedicina Cima, S.L. Compositions based on the fibronectin extracellular domain a for the treatment of melanoma
US8685416B2 (en) 2010-03-02 2014-04-01 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Compositions and methods for the treatment of cancer
EP2542670A2 (en) 2010-03-05 2013-01-09 President and Fellows of Harvard College Induced dendritic cell compositions and uses thereof
GB201003920D0 (en) 2010-03-09 2010-04-21 Glaxosmithkline Biolog Sa Method of treatment
GB201003924D0 (en) 2010-03-09 2010-04-21 Glaxosmithkline Biolog Sa Immunogenic composition
JP2013521327A (en) 2010-03-10 2013-06-10 グラクソスミスクライン バイオロジカルズ ソシエテ アノニム Immunogenic composition
WO2011112599A2 (en) 2010-03-12 2011-09-15 The United States Of America, As Represented By The Secretary. Department Of Health & Human Services Immunogenic pote peptides and methods of use
EP2547327B1 (en) 2010-03-19 2018-09-05 Massachusetts Institute of Technology Lipid vesicle compositions and methods of use
US20110229556A1 (en) * 2010-03-19 2011-09-22 Massachusetts Institute Of Technology Lipid-coated polymer particles for immune stimulation
US9149432B2 (en) 2010-03-19 2015-10-06 Massachusetts Institute Of Technology Lipid vesicle compositions and methods of use
GB201005005D0 (en) 2010-03-25 2010-05-12 Angeletti P Ist Richerche Bio New vaccine
ES2910199T3 (en) 2010-03-30 2022-05-11 Childrens Hospital & Res Center At Oakland Factor H-binding proteins (fHbp) with altered properties and methods of using them
BR112012026227A2 (en) 2010-04-13 2020-08-04 Celldex Therapeutics, Inc. human or humanized monoclonal antibody, bispecific molecule, expression vector, transformed cell, composition, and, uses of an antibody
WO2011139709A2 (en) 2010-04-26 2011-11-10 Chimerix, Inc. Methods of treating retroviral infections and related dosage regimes
DK2569436T3 (en) 2010-05-14 2018-03-05 Univ Oregon Health & Science RECOMBINANT HCMV AND RHCMV VECTORS AND APPLICATIONS THEREOF
KR20130108987A (en) 2010-05-26 2013-10-07 셀렉타 바이오사이언시즈, 인크. Multivalent synthetic nanocarrier vaccines
ES2683316T3 (en) 2010-05-28 2018-09-26 Zoetis Belgium S.A. Vaccines that comprise cholesterol and CpG as the only adjuvant transporter molecules
WO2011154878A1 (en) 2010-06-07 2011-12-15 Pfizer Vaccines Llc Ige ch3 peptide vaccine
US8658603B2 (en) 2010-06-16 2014-02-25 The Regents Of The University Of Michigan Compositions and methods for inducing an immune response
KR101761388B1 (en) 2010-07-30 2017-07-25 큐어백 아게 Complexation of nucleic acids with disulfide-crosslinked cationic components for transfection and immunostimulation
CN103957931B (en) 2010-09-03 2017-10-24 瓦尔内瓦奥地利有限责任公司 Isolated polypeptide of clostridium difficile toxin A and toxin B albumen and application thereof
WO2012031760A1 (en) 2010-09-08 2012-03-15 Medigene Ag Parvovirus mutated structural proteins comprising cross - protective b - cell epitopes of a hpv l2 protein as well as products and methods relating thereto
GB201015132D0 (en) 2010-09-10 2010-10-27 Univ Bristol Vaccine composition
CA2811103C (en) 2010-09-14 2020-01-14 University Of Pittsburgh-Of The Commonwealth System Of Higher Education Computationally optimized broadly reactive antigens for influenza
EP2620428B1 (en) 2010-09-24 2019-05-22 Wave Life Sciences Ltd. Asymmetric auxiliary group
MX354752B (en) 2010-09-27 2018-03-20 Janssen Vaccines & Prevention Bv Heterologous prime boost vaccination regimen against malaria.
LT2624873T (en) 2010-10-06 2020-03-10 President And Fellows Of Harvard College Injectable, pore-forming hydrogels for materials-based cell therapies
US20130345079A1 (en) 2010-10-27 2013-12-26 Infectious Disease Research Institute Mycobacterium tuberculosis antigens and combinations thereof having high seroreactivity
EP2640190A4 (en) 2010-11-05 2016-05-11 Selecta Biosciences Inc Modified nicotinic compounds and related methods
ES2859673T3 (en) 2010-11-08 2021-10-04 Infectious Disease Res Inst Vaccines comprising nonspecific nucleoside hydrolase polypeptides and sterol 24-c-methyltransferase (SMT) for the treatment and diagnosis of leishmaniasis
CN106822882A (en) 2010-12-14 2017-06-13 葛兰素史密丝克莱恩生物有限公司 Mycobacterial antigen composition
EP2667891B1 (en) 2011-01-27 2021-10-06 Gamma Vaccines Pty Limited Combination vaccines
CA2828068C (en) 2011-02-22 2019-03-19 Biondvax Pharmaceuticals Ltd. Multimeric multiepitope polypeptides in improved seasonal and pandemic influenza vaccines
US20140004142A1 (en) 2011-03-02 2014-01-02 Pfizer Inc. Pcsk9 vaccine
EP2505640A1 (en) 2011-03-29 2012-10-03 Neo Virnatech, S.L. Vaccine compositions for birnavirus-borne diseases
GB201106357D0 (en) 2011-04-14 2011-06-01 Pessi Antonello Composition and uses thereof
AU2012243039B2 (en) 2011-04-08 2017-07-13 Immune Design Corp. Immunogenic compositions and methods of using the compositions for inducing humoral and cellular immune responses
TW201302779A (en) 2011-04-13 2013-01-16 Glaxosmithkline Biolog Sa Fusion proteins & combination vaccines
WO2012149307A2 (en) 2011-04-27 2012-11-01 Immune Design Corp. Synthetic long peptide (slp)-based vaccines
FR2975600B1 (en) 2011-05-24 2013-07-05 Assist Publ Hopitaux De Paris AGENTS FOR THE TREATMENT OF TUMORS
CN103717070A (en) 2011-06-01 2014-04-09 贾纳斯生物治疗有限公司 Novel immune system modulators
US9353115B2 (en) 2011-06-01 2016-05-31 Janus Biotherapeutics, Inc. Immune system modulators
EP3153180A1 (en) 2011-06-03 2017-04-12 3M Innovative Properties Company Heterobifunctional linkers with polyethylene glycol segments and immune response modifier conjugates made therefrom
CN103582496B (en) 2011-06-03 2016-05-11 3M创新有限公司 There is the Heterobifunctional connection base of polyethylene glycol segment and the immune response modifier conjugate of being made by it
WO2012170356A1 (en) 2011-06-04 2012-12-13 Rochester General Hospital Research Institute Compositions and methods related to p6 of haemophilus influenzae
CA2839932C (en) 2011-06-07 2022-07-05 Ibio, Inc. In vivo de-glycosylation of recombinant proteins by co-expression with pngase f
AU2012271336B2 (en) 2011-06-17 2017-03-02 University Of Tennessee Research Foundation Group A streptococcus multivalent vaccine
BR112013032723A2 (en) 2011-06-20 2017-01-31 Univ Pittsburgh Commonwealth Sys Higher Education widely reactive antigens computationally optimized for influenza h1n1
CN103608030A (en) 2011-06-21 2014-02-26 昂科发克特公司 Compositions and methods for therapy and diagnosis of cancer
WO2013007703A1 (en) * 2011-07-08 2013-01-17 Universität Zürich CLASS A CpG OLIGONUCLEOTIDES FOR PREVENTION OF VIRAL INFECTION IN CATS
KR20140068884A (en) 2011-07-19 2014-06-09 웨이브 라이프 사이언시스 피티이. 리미티드 Methods for the synthesis of functionalized nucleic acids
US20130023736A1 (en) 2011-07-21 2013-01-24 Stanley Dale Harpstead Systems for drug delivery and monitoring
CN109172819A (en) 2011-07-29 2019-01-11 西莱克塔生物科技公司 Generate the synthesis nano-carrier of body fluid and cytotoxic T lymphocyte (CTL) immune response
GB201114919D0 (en) 2011-08-30 2011-10-12 Glaxosmithkline Biolog Sa Method
WO2013039792A1 (en) 2011-09-12 2013-03-21 The United States Of America As Represented By The Secretary, Department Of Health And Human Services Immunogens based on an hiv-1 gp120 v1v2 epitope
US10752676B2 (en) 2011-09-16 2020-08-25 Ucb Biopharma Sprl Neutralising antibodies to the major exotoxins TCDA and TCDB of Clostridium difficile
EP2572725B1 (en) 2011-09-21 2015-07-08 Ruprecht-Karls-Universität Heidelberg MSI-specific frameshift peptides (FSP) for prevention and treatment of cancer
US20140234360A1 (en) 2011-09-30 2014-08-21 The United States of America, as represented by the Secretary, Dept.of Health and Human Services Influenza vaccine
JP2014528449A (en) 2011-10-04 2014-10-27 ジャナス バイオセラピューティクス，インク． Novel imidazole quinoline immune system modulator
EP2768847A4 (en) 2011-10-20 2015-11-11 Us Gov Health & Human Serv Dengue virus e-glycoprotein polypeptides containing mutations that eliminate immunodominant cross-reactive epitopes
WO2013074501A1 (en) 2011-11-14 2013-05-23 Crucell Holland B.V. Heterologous prime-boost immunization using measles virus-based vaccines
EP2596806A1 (en) 2011-11-25 2013-05-29 Index Pharmaceuticals AB Method for prevention of colectomy
SG10201605350UA (en) 2012-01-16 2016-08-30 Mckenna Elizabeth Compositions and Methods for the Treatment of Hepatic Diseases and Disorders
US20140086849A1 (en) 2012-09-21 2014-03-27 Elizabeth McKenna Naturally-Occurring CpG Oligonucleotide Compositions and Therapeutic Applications Thereof
WO2013113326A1 (en) 2012-01-31 2013-08-08 Curevac Gmbh Pharmaceutical composition comprising a polymeric carrier cargo complex and at least one protein or peptide antigen
EP2811981B1 (en) 2012-02-07 2019-05-08 Infectious Disease Research Institute Improved adjuvant formulations comprising tlr4 agonists and methods of using the same
RU2653756C2 (en) 2012-02-07 2018-05-14 Юниверсити Оф Питтсбург - Оф Зе Коммонвэлс Систем Оф Хайе Эдьюкейшн Computationally optimized broadly reactive antigens for h3n2 viruses
SG11201404147YA (en) 2012-02-13 2014-10-30 Univ Pittsburgh Computationally optimized broadly reactive antigens for human and avian h5n1 influenza
CA2866185C (en) 2012-03-23 2021-04-06 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Pathogenic phlebovirus isolates and compositions and methods of use
EP3492095A1 (en) 2012-04-01 2019-06-05 Technion Research & Development Foundation Limited Extracellular matrix metalloproteinase inducer (emmprin) peptides and binding antibodies
ES2773895T3 (en) 2012-04-16 2020-07-15 Harvard College Mesoporous Silica Compositions to Modulate Immune Responses
EP2841098A4 (en) 2012-04-23 2016-03-02 Allertein Therapeutics Llc Nanoparticles for treatment of allergy
EP2844282B1 (en) 2012-05-04 2019-06-12 Pfizer Inc Prostate-associated antigens and vaccine-based immunotherapy regimens
ES2787455T3 (en) 2012-05-16 2020-10-16 Immune Design Corp HSV-2 vaccines
IN2014DN09618A (en) 2012-05-23 2015-07-31 Us Sec Dep Of Health And Human Services
EP2666785A1 (en) 2012-05-23 2013-11-27 Affiris AG Complement component C5a-based vaccine
CN104428008B (en) 2012-05-24 2020-10-09 美国政府(由卫生和人类服务部的部长所代表) Multivalent meningococcal conjugates and methods of making conjugates
US9687542B2 (en) 2012-06-19 2017-06-27 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Rift valley fever virus replicon particles and use thereof
MX356830B (en) 2012-07-13 2018-06-15 Shin Nippon Biomedical Laboratories Ltd Chiral nucleic acid adjuvant.
JP6268157B2 (en) 2012-07-13 2018-01-24 株式会社Ｗａｖｅ Ｌｉｆｅ Ｓｃｉｅｎｃｅｓ Ｊａｐａｎ Asymmetric auxiliary group
CN112007045A (en) 2012-07-13 2020-12-01 波涛生命科学有限公司 Chiral control
RU2659149C2 (en) 2012-08-03 2018-06-28 Инфекшес Дизиз Рисерч Инститьют Compositions and methods for treating active mycobacterium tuberculosis infection
US20140037680A1 (en) 2012-08-06 2014-02-06 Glaxosmithkline Biologicals, S.A. Novel method
CA2879939A1 (en) 2012-08-06 2014-02-13 Glaxosmithkline Biologicals S.A. Novel method
US9605276B2 (en) 2012-08-24 2017-03-28 Etubics Corporation Replication defective adenovirus vector in vaccination
EP2703483A1 (en) 2012-08-29 2014-03-05 Affiris AG PCSK9 peptide vaccine
WO2014037124A1 (en) 2012-09-04 2014-03-13 Bavarian Nordic A/S Methods and compositions for enhancing vaccine immune responses
WO2014043518A1 (en) 2012-09-14 2014-03-20 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Brachyury protein, non-poxvirus non-yeast vectors encoding brachyury protein, and their use
US10232035B2 (en) 2012-09-14 2019-03-19 The Regents Of The University Of Colorado, A Body Corporate Conditionally replication deficient herpes virus and use thereof in vaccines
WO2014043535A1 (en) 2012-09-14 2014-03-20 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Compositions for the treatment of cancer
JP2016501515A (en) 2012-10-24 2016-01-21 プレートレット ターゲテッド セラピューティクス，エルエルシー Platelet targeted therapy
RU2644677C2 (en) 2012-12-13 2018-02-13 Рупрехт-Карлс-Университэт Гейдельберг Msi-specific peptides with shifted reading frame (srf) for prevention and treatment of cancer
DK2941233T3 (en) 2013-01-07 2020-10-19 Univ Pennsylvania Compositions and methods for treating cutaneous T-cell lymphoma
JP2016507520A (en) 2013-01-23 2016-03-10 ザ ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティー Stabilized hepatitis B core polypeptide
WO2014160463A1 (en) 2013-03-13 2014-10-02 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Prefusion rsv f proteins and their use
US10058603B2 (en) 2013-03-15 2018-08-28 Glaxosmithkline Biologicals S.A. Vaccine
WO2014160987A2 (en) 2013-03-28 2014-10-02 Infectious Disease Research Institute Vaccines comprising leishmania polypeptides for the treatment and diagnosis of leishmaniasis
WO2014165679A1 (en) 2013-04-03 2014-10-09 Allertein Therapeutics, Llc Novel nanoparticle compositions
US8957047B2 (en) 2013-04-18 2015-02-17 Immune Design Corp. GLA monotherapy for use in cancer treatment
CN105392882A (en) 2013-04-19 2016-03-09 加利福尼亚大学董事会 Lone star virus
WO2015132619A1 (en) 2013-05-15 2015-09-11 The Governors Of The University Of Alberta E1e2 hcv vaccines and methods of use
US9463198B2 (en) 2013-06-04 2016-10-11 Infectious Disease Research Institute Compositions and methods for reducing or preventing metastasis
GB201310008D0 (en) 2013-06-05 2013-07-17 Glaxosmithkline Biolog Sa Immunogenic composition for use in therapy
WO2014201245A1 (en) 2013-06-12 2014-12-18 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Tlr-9 agonist with tlr-7 and/or tlr-8 agonist for treating tumors
JP6697384B2 (en) 2013-07-25 2020-05-20 イグジキュア, インコーポレーテッドExicure, Inc. Spherical nucleic acid-based constructs as immunostimulants for prophylactic and therapeutic use
WO2015011254A1 (en) 2013-07-26 2015-01-29 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods and pharmaceutical compositions for the treatment of bacterial infections
JP6564367B2 (en) 2013-08-05 2019-08-21 グラクソスミスクライン バイオロジカルズ ソシエテ アノニム Combined immunogenic composition
SG11201510746WA (en) 2013-08-21 2016-03-30 Curevac Ag Respiratory syncytial virus (rsv) vaccine
US9603944B2 (en) 2013-09-27 2017-03-28 Massachusetts Institute Of Technology Carrier-free biologically-active protein nanostructures
ES2957209T3 (en) 2013-09-30 2024-01-15 Triad Nat Security Llc Conserved region HIV mosaic immunogenic polypeptides
AU2014329393B2 (en) 2013-10-04 2020-04-30 Pin Pharma, Inc. Immunostimulatory HIV Tat derivative polypeptides for use in cancer treatment
DK3062815T3 (en) 2013-11-01 2019-03-11 Pfizer Vectors for expression of prostate-associated antigens
CA2928700C (en) 2013-11-01 2019-01-15 University Of Oslo Albumin variants and uses thereof
US10357554B2 (en) 2013-11-11 2019-07-23 The United States Of America, As Represented By The Secretary Of The Army AMA-1 epitopes, antibodies, compositions, and methods of making and using the same
EP3068426B1 (en) 2013-11-13 2020-02-12 University Of Oslo Outer membrane vesicles and uses thereof
US11452767B2 (en) 2013-11-15 2022-09-27 Oslo Universitetssykehus Hf CTL peptide epitopes and antigen-specific t cells, methods for their discovery, and uses thereof
US20160287696A1 (en) 2013-11-20 2016-10-06 La Jolla Institute For Allergy And Immunology Pan pollen immunogens and methods and uses for immune response modulation
WO2015077442A2 (en) 2013-11-20 2015-05-28 La Jolla Institute For Allergy And Immunology Grass pollen immunogens and methods and uses for immune response modulation
NZ719934A (en) 2013-11-28 2022-10-28 Bavarian Nordic As Compositions and methods vectors for inducing an enhanced immune response using poxvirus vectors
JP6758185B2 (en) 2013-12-13 2020-09-23 ザ ユナイテッド ステイツ オブ アメリカ， アズ リプレゼンテッド バイ ザ セクレタリー， デパートメント オブ ヘルス アンド ヒューマン サービシーズ Multiepitope TARP peptide vaccine and its use
WO2015092710A1 (en) 2013-12-19 2015-06-25 Glaxosmithkline Biologicals, S.A. Contralateral co-administration of vaccines
IL246456B2 (en) 2013-12-31 2024-06-01 Access To Advanced Health Inst Single vial vaccine formulations
WO2015108048A1 (en) * 2014-01-15 2015-07-23 株式会社新日本科学 Chiral nucleic acid adjuvant having antitumor effect and antitumor agent
EP3095460A4 (en) 2014-01-15 2017-08-23 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant having anti-allergic activity, and anti-allergic agent
JPWO2015108047A1 (en) * 2014-01-15 2017-03-23 株式会社新日本科学 Chiral nucleic acid adjuvant having immunity induction activity and immunity induction activator
PL3094728T3 (en) 2014-01-16 2022-07-04 Wave Life Sciences Ltd. Chiral design
CA2937184A1 (en) 2014-01-21 2015-07-30 Pfizer Inc. Streptococcus pneumoniae capsular polysaccharides and conjugates thereof
ES2820824T3 (en) 2014-01-21 2021-04-22 Pfizer Immunogenic Compositions Comprising Conjugated Capsular Saccharide Antigens and Uses Thereof
US11160855B2 (en) 2014-01-21 2021-11-02 Pfizer Inc. Immunogenic compositions comprising conjugated capsular saccharide antigens and uses thereof
EA201691452A1 (en) 2014-01-21 2016-12-30 Иммьюн Дизайн Корп. COMPOSITIONS FOR APPLICATION IN THE TREATMENT OF ALLERGIC STATES
JP6585624B2 (en) 2014-01-21 2019-10-02 ファイザー・インク Streptococcus pneumoniae capsular polysaccharides and conjugates thereof
EP3104877B1 (en) 2014-02-11 2020-01-22 The USA, as represented by The Secretary, Department of Health and Human Services Pcsk9 vaccine and methods of using the same
TW201620927A (en) 2014-02-24 2016-06-16 葛蘭素史密斯克藍生物品公司 USPA2 protein constructs and uses thereof
WO2015131053A1 (en) 2014-02-28 2015-09-03 Alk-Abelló A/S Polypeptides derived from phl p and methods and uses thereof for immune response modulation
US10369216B2 (en) 2014-04-01 2019-08-06 Curevac Ag Polymeric carrier cargo complex for use as an immunostimulating agent or as an adjuvant
EP3134104B1 (en) 2014-04-25 2018-12-26 Tria Bioscience Corp. Synthetic hapten carrier compositions and methods
EP3137105A4 (en) 2014-04-30 2017-12-27 President and Fellows of Harvard College Combination vaccine devices and methods of killing cancer cells
US20170121373A1 (en) 2014-05-30 2017-05-04 Sanofi Pasteur Inc. Expression and conformational analysis of engineered influenza hemagglutinin
JP6581604B2 (en) 2014-06-04 2019-09-25 イグジキュア, インコーポレーテッドExicure, Inc. Multivalent delivery of immunomodulators with liposomal globular nucleic acids for prophylactic or therapeutic applications
EP2952893A1 (en) 2014-06-04 2015-12-09 Institut d'Investigació Biomèdica de Bellvitge (IDIBELL) Method for detecting antibody-secreting B cells specific for HLA
EP3888676A1 (en) 2014-06-13 2021-10-06 GlaxoSmithKline Biologicals S.A. Immunogenic combinations
EP3169352A1 (en) 2014-07-15 2017-05-24 Immune Design Corp. Prime-boost regimens with a tlr4 agonist adjuvant and a lentiviral vector
CN106715458A (en) 2014-07-18 2017-05-24 华盛顿大学 Cancer vaccine compositions and methods of use thereof
WO2016012385A1 (en) 2014-07-21 2016-01-28 Sanofi Pasteur Vaccine composition comprising ipv and cyclodextrins
JP2017525711A (en) 2014-08-22 2017-09-07 ジャナス バイオセラピューティクス，インク． Novel N2, N4, N7,6-tetrasubstituted pteridine-2,4,7-triamine and 2,4,6,7-tetrasubstituted pteridine compounds and methods for their synthesis and use
EP3204499A4 (en) 2014-10-06 2018-04-25 Exicure, Inc. Anti-tnf compounds
US11083788B2 (en) 2014-10-10 2021-08-10 The Regents Of The University Of Michigan Nanoemulsion compositions for preventing, suppressing or eliminating allergic and inflammatory disease
AR102548A1 (en) 2014-11-07 2017-03-08 Takeda Vaccines Inc VACCINES AGAINST HAND, FEET AND MOUTH DISEASE AND MANUFACTURING AND USE METHODS
MA40920A (en) 2014-11-07 2017-09-12 Takeda Vaccines Inc VACCINES OF THE HAND, FOOT AND MOUTH, AND METHODS OF MANUFACTURING AND USE THEREOF
WO2016081911A2 (en) 2014-11-21 2016-05-26 Northwestern University The sequence-specific cellular uptake of spherical nucleic acid nanoparticle conjugates
AU2015359503B2 (en) 2014-12-10 2019-05-09 Glaxosmithkline Biologicals Sa Method of treatment
ES2881346T3 (en) 2014-12-19 2021-11-29 Regenesance B V Antibodies that bind to human C6 and uses thereof
MX2017008658A (en) 2014-12-31 2018-02-23 Checkmate Pharmaceuticals Inc Combination tumor immunotherapy.
US11352642B2 (en) 2015-01-09 2022-06-07 Etubics Corporation Methods and compositions for combination immunotherapy
HRP20230416T1 (en) 2015-01-15 2023-07-07 Pfizer Inc. Immunogenic compositions for use in pneumococcal vaccines
CA3012602A1 (en) 2015-01-30 2016-08-04 President And Fellows Of Harvard College Peritumoral and intratumoral materials for cancer therapy
US20180036334A1 (en) 2015-02-13 2018-02-08 Icahn School Of Medicine At Mount Sinai Rna containing compositions and methods of their use
WO2016140702A1 (en) 2015-03-03 2016-09-09 The United States Of America, As Represented By The Secretary, Department Of Health & Human Serivces Display platform from bacterial spore coat proteins
EP4226937A3 (en) 2015-03-05 2023-09-27 Northwestern University Non-neuroinvasive viruses and uses thereof
WO2016145085A2 (en) 2015-03-09 2016-09-15 Celldex Therapeutics, Inc. Cd27 agonists
JP2018511655A (en) 2015-03-20 2018-04-26 ザ リージェンツ オブ ザ ユニバーシティ オブ ミシガン Immunogenic compositions for use in vaccination against Bordetella
WO2016149771A1 (en) 2015-03-26 2016-09-29 Gamma Vaccines Pty Limited Streptococcal vaccine
CN114099793A (en) 2015-04-10 2022-03-01 哈佛学院院长等 Immune cell capture device and methods of making and using same
US11149087B2 (en) 2015-04-20 2021-10-19 Etubics Corporation Methods and compositions for combination immunotherapy
WO2016180852A1 (en) 2015-05-12 2016-11-17 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for preparing antigen-specific t cells from an umbilical cord blood sample
MA42420A (en) 2015-05-13 2018-05-23 Agenus Inc VACCINES FOR THE TREATMENT AND PREVENTION OF CANCER
MX2017015004A (en) 2015-05-26 2018-07-06 Ohio State Innovation Foundation Nanoparticle based vaccine strategy against swine influenza virus.
AU2016270979B2 (en) 2015-06-02 2020-11-12 Sanofi Pasteur Inc. Engineered influenza antigenic polypeptides and immunogenic compositions thereof
US10954492B2 (en) 2015-06-10 2021-03-23 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Processes for production and purification of nucleic acid-containing compositions
US20190233825A1 (en) * 2015-06-26 2019-08-01 Bayer Animal Health Gmbh Methods of modulating cytosolic dna surveillance molecules
SG10202005253TA (en) 2015-07-21 2020-07-29 Pfizer Immunogenic compositions comprising conjugated capsular saccharide antigens, kits comprising the same and uses thereof
CA2995212A1 (en) 2015-08-12 2017-02-16 Massachusetts Institute Of Technology Cell surface coupling of nanoparticles
CN108024914A (en) 2015-09-17 2018-05-11 Jrx生物技术有限公司 Improve the method for the aquation or wetting action of skin
WO2017059280A1 (en) 2015-10-02 2017-04-06 The University Of North Carolina At Chapel Hill Novel pan-tam inhibitors and mer/axl dual inhibitors
US10548970B2 (en) 2015-10-05 2020-02-04 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Human rotavirus G9P[6] strain and use as a vaccine
GB201518668D0 (en) 2015-10-21 2015-12-02 Glaxosmithkline Biolog Sa Immunogenic Comosition
US11266726B2 (en) 2015-10-30 2022-03-08 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Compositions and methods for the treatment of HER2-expressing solid tumors
EP3377098A1 (en) 2015-11-20 2018-09-26 Pfizer Inc Immunogenic compositions for use in pneumococcal vaccines
WO2017120504A1 (en) 2016-01-08 2017-07-13 Durfee Paul N Osteotropic nanoparticles for prevention or treatment of bone metastases
PL3407910T3 (en) 2016-01-29 2022-08-16 Bavarian Nordic A/S Recombinant modified vaccinia virus ankara (mva) equine encephalitis virus vaccine
WO2017136837A1 (en) 2016-02-06 2017-08-10 President And Fellows Of Harvard College Recapitulating the hematopoietic niche to reconstitute immunity
EP3426289A4 (en) 2016-03-10 2019-08-07 Aperisys, Inc. Antigen-binding fusion proteins with modified hsp70 domains
MY195541A (en) 2016-03-14 2023-01-31 Univ Oslo Engineered Immunoglobulins With Altered Fcrn Binding
WO2017158421A1 (en) 2016-03-14 2017-09-21 University Of Oslo Anti-viral engineered immunoglobulins
EP3436066A1 (en) 2016-04-01 2019-02-06 Checkmate Pharmaceuticals, Inc. Fc receptor-mediated drug delivery
BR112018071307A2 (en) 2016-04-18 2019-02-26 Celldex Therapeutics, Inc. human cd40 binding agonist antibodies and uses thereof
WO2017189448A1 (en) 2016-04-25 2017-11-02 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Bivalent immunogenic conjugate for malaria and typhoid
CN109310773B (en) 2016-05-16 2022-04-26 传染病研究所 Formulations containing TLR agonists and methods of use
US11173207B2 (en) 2016-05-19 2021-11-16 The Regents Of The University Of Michigan Adjuvant compositions
EP3458088A2 (en) 2016-05-21 2019-03-27 Infectious Disease Research Institute Compositions and methods for treating secondary tuberculosis and nontuberculous mycobacterium infections
KR20190034504A (en) 2016-05-27 2019-04-02 이투빅스 코포레이션 Neo-epitope vaccine compositions and methods for their use
IL311086A (en) 2016-06-01 2024-04-01 Access To Advanced Health Inst Nanoalum particles containing a sizing agent
US10844097B2 (en) 2016-06-02 2020-11-24 Sanofi Pasteur Inc. Engineered influenza antigenic polypeptides and immunogenic compositions thereof
SG11201810758WA (en) 2016-06-03 2018-12-28 Sanofi Pasteur Inc Modification of engineered influenza hemagglutinin polypeptides
CA3026807A1 (en) 2016-06-13 2017-12-21 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Nucleic acids encoding zika virus-like particles and their use in zika virus vaccines and diagnostic assays
CN109715196A (en) 2016-06-13 2019-05-03 转矩医疗股份有限公司 For promoting the composition and method of immune cell function
GB201610599D0 (en) 2016-06-17 2016-08-03 Glaxosmithkline Biologicals Sa Immunogenic Composition
EP3471761A2 (en) 2016-06-21 2019-04-24 University Of Oslo Hla binding vaccine moieties and uses thereof
WO2018009603A1 (en) 2016-07-08 2018-01-11 The United State of America, as represented by the Secretary, Department of Health and Human Service Chimeric west nile/zika viruses and methods of use
WO2018009604A1 (en) 2016-07-08 2018-01-11 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Chimeric dengue/zika viruses live-attenuated zika virus vaccines
JP2019522486A (en) 2016-07-13 2019-08-15 プレジデント アンド フェローズ オブ ハーバード カレッジ Antigen presenting cell mimetic scaffold and methods for making and using the same
TWI789357B (en) 2016-08-05 2023-01-11 南韓商Ｓｋ生物科技股份有限公司 Multivalent pneumococcal polysaccharide-protein conjugate composition
SG11201900789VA (en) 2016-08-05 2019-02-27 Sanofi Pasteur Inc Multivalent pneumococcal polysaccharide-protein conjugate composition
BR112019003462A2 (en) 2016-08-23 2019-06-25 Glaxosmithkline Biologicals Sa fusion protein, polynucleotide, viral vector, pharmaceutical composition, use of a fusion protein, polynucleotide, viral vector or pharmaceutical composition, and method for treating or preventing viral hepatitis b infection or viral hepatitis c infection
US11364304B2 (en) 2016-08-25 2022-06-21 Northwestern University Crosslinked micellar spherical nucleic acids
MX2019003035A (en) 2016-09-16 2019-09-13 Infectious Disease Res Inst Vaccines comprising mycobacterium leprae polypeptides for the prevention, treatment, and diagnosis of leprosy.
BE1024774B1 (en) 2016-09-29 2018-07-02 Glaxosmithkline Biologicals Sa COMPOSITIONS AND METHODS OF TREATMENT
US11959083B2 (en) * 2016-10-07 2024-04-16 Secarna Pharmaceuticals Gmbh & Co. Kg Immunosuppression-reverting oligonucleotides inhibiting the expression of CD39
US10172933B2 (en) 2016-10-31 2019-01-08 The United States Of America, As Represented By The Secretary Of Agriculture Mosaic vaccines for serotype a foot-and-mouth disease virus
WO2018096396A1 (en) 2016-11-22 2018-05-31 University Of Oslo Albumin variants and uses thereof
GB201620968D0 (en) 2016-12-09 2017-01-25 Glaxosmithkline Biologicals Sa Adenovirus polynucleotides and polypeptides
US11084850B2 (en) 2016-12-16 2021-08-10 The Pirbright Institute Recombinant prefusion RSV F proteins and uses thereof
HUE059252T2 (en) 2017-01-20 2022-11-28 Pfizer Immunogenic compositions for use in pneumococcal vaccines
WO2018160865A1 (en) 2017-03-01 2018-09-07 Charles Jeffrey Brinker Active targeting of cells by monosized protocells
WO2018162450A1 (en) 2017-03-06 2018-09-13 Fundación Para La Investigación Médica Aplicada New inmunostimulatory compositions comprising an entity of cold inducible rna-binding protein with an antigen for the activation of dendritic cells
EP3600391A1 (en) 2017-03-31 2020-02-05 GlaxoSmithKline Intellectual Property Development Limited Immunogenic composition, use and method of treatment
WO2018178265A1 (en) 2017-03-31 2018-10-04 Glaxosmithkline Intellectual Property Development Limited Immunogenic composition, use and method of treatment
CN118085055A (en) 2017-04-19 2024-05-28 生物医学研究所 Plasmodium sporozoite NPDP peptides as vaccine and novel malaria vaccine and antibody binding targets
US11696954B2 (en) 2017-04-28 2023-07-11 Exicure Operating Company Synthesis of spherical nucleic acids using lipophilic moieties
SG11201911930SA (en) 2017-06-11 2020-01-30 Molecular Express Inc Methods and compositions for substance use disorder vaccine formulations and uses thereof
WO2018232257A1 (en) 2017-06-15 2018-12-20 Infectious Disease Research Institute Nanostructured lipid carriers and stable emulsions and uses thereof
WO2019018744A1 (en) 2017-07-21 2019-01-24 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Neisseria meningitidis immunogenic compositions
WO2019034575A1 (en) 2017-08-14 2019-02-21 Glaxosmithkline Biologicals Sa Methods of boosting immune responses
US11123415B2 (en) 2017-08-16 2021-09-21 Ohio State Innovation Foundation Nanoparticle compositions for Salmonella vaccines
CA3074826A1 (en) 2017-09-05 2019-03-14 Torque Therapeutics, Inc. Therapeutic protein compositions and methods of making and using the same
EP3678698A1 (en) 2017-09-07 2020-07-15 University Of Oslo Vaccine molecules
EP3678699A1 (en) 2017-09-07 2020-07-15 University Of Oslo Vaccine molecules
WO2019051149A1 (en) 2017-09-08 2019-03-14 Infectious Disease Research Institute Liposomal formulations comprising saponin and methods of use
EP4183882A1 (en) 2017-09-08 2023-05-24 MiNA Therapeutics Limited Stabilized hnf4a sarna compositions and methods of use
WO2019089755A1 (en) 2017-10-31 2019-05-09 Western Oncolytics Ltd. Platform oncolytic vector for systemic delivery
CN111511395B (en) 2017-11-03 2024-10-15 武田疫苗股份有限公司 Methods for inactivating Zika virus and for determining completeness of inactivation
EP3703745B1 (en) 2017-11-04 2024-04-10 Nevada Research & Innovation Corporation Immunogenic conjugates and methods of use thereof
JP7458977B2 (en) 2017-12-15 2024-04-01 エランコ アニマル ヘルス ゲー・エム・ベー・ハー Immunostimulatory Compositions
US11116830B2 (en) 2017-12-18 2021-09-14 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Bacterial polysaccharide-conjugated carrier proteins and use thereof
EP3727447A1 (en) 2017-12-19 2020-10-28 Massachusetts Institute of Technology Antigen-adjuvant coupling reagents and methods of use
GB201721582D0 (en) 2017-12-21 2018-02-07 Glaxosmithkline Biologicals Sa S aureus antigens and immunogenic compositions
GB201721576D0 (en) 2017-12-21 2018-02-07 Glaxosmithkline Biologicals Sa Hla antigens and glycoconjugates thereof
US11369675B2 (en) 2018-01-22 2022-06-28 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Broadly protective inactivated influenza virus vaccine
CN111836639A (en) 2018-01-26 2020-10-27 河谷细胞有限公司 Compositions and methods for combination cancer vaccine and immune adjuvant therapy
CN112074295A (en) 2018-02-16 2020-12-11 2A制药公司 Parvovirus structural proteins for the treatment of autoimmune diseases
EP3527223A1 (en) 2018-02-16 2019-08-21 2A Pharma AB Mutated parvovirus structural protein
EP3759129A1 (en) 2018-02-28 2021-01-06 Pfizer Inc Il-15 variants and uses thereof
WO2019173438A1 (en) 2018-03-06 2019-09-12 Stc. Unm Compositions and methods for reducing serum triglycerides
CN112189019B (en) 2018-03-28 2024-10-25 圣诺菲·帕斯图尔公司 Methods of producing broadly protective vaccine compositions comprising hemagglutinin
WO2019195276A1 (en) 2018-04-03 2019-10-10 Sanofi Antigenic ospa polypeptides
WO2019195314A2 (en) 2018-04-03 2019-10-10 Sanofi Antigenic epstein barr virus polypeptides
WO2019195291A1 (en) 2018-04-03 2019-10-10 Sanofi Antigenic respiratory syncytial virus polypeptides
EP3773703A1 (en) 2018-04-03 2021-02-17 Sanofi Antigenic influenza-ferritin polypeptides
EP3773698A1 (en) 2018-04-03 2021-02-17 Sanofi Ferritin proteins
EP3775218A1 (en) 2018-04-09 2021-02-17 Checkmate Pharmaceuticals Packaging oligonucleotides into virus-like particles
CA3097369A1 (en) 2018-04-17 2019-10-24 Celldex Therapeutics, Inc. Anti-cd27 and anti-pd-l1 antibodies and bispecific constructs
TWI816396B (en) 2018-05-23 2023-09-21 美商輝瑞大藥廠 Antibodies specific for gucy2c and uses thereof
KR102602329B1 (en) 2018-05-23 2023-11-16 화이자 인코포레이티드 Antibodies specific for CD3 and their uses
CA3102224A1 (en) 2018-06-12 2019-12-19 Glaxosmithkline Biologicals Sa Chimpanzee adenovirus chad157 polynucleotides, polypeptides.vectors, compositions, cells and uses thereof
CA3106291A1 (en) 2018-07-19 2020-01-23 Glaxosmithkline Biologicals Sa Processes for preparing dried polysaccharides
BR112021000965A2 (en) 2018-08-07 2021-04-27 Glaxosmithkline Biologicals S.A. processes and vaccines
US11260119B2 (en) 2018-08-24 2022-03-01 Pfizer Inc. Escherichia coli compositions and methods thereof
US20220118077A1 (en) 2018-09-11 2022-04-21 Shanghai Public Health Clinical Center Immunogen for broad-spectrum influenza vaccine and application thereof
CN113454100B (en) 2018-12-04 2024-08-23 洛克菲勒大学 HIV vaccine immunogens
WO2020115171A1 (en) 2018-12-06 2020-06-11 Glaxosmithkline Biologicals Sa Immunogenic compositions
EP3893926A1 (en) 2018-12-12 2021-10-20 Pfizer Inc. Immunogenic multiple hetero-antigen polysaccharide-protein conjugates and uses thereof
EP3894431A2 (en) 2018-12-12 2021-10-20 GlaxoSmithKline Biologicals SA Modified carrier proteins for o-linked glycosylation
US20220023413A1 (en) 2018-12-12 2022-01-27 The U.S.A., As Represented By The Secretary, Department Of Health And Human Services Recombinant mumps virus vaccine expressing genotype g fusion and hemagglutinin-neuraminidase proteins
US20220023410A1 (en) 2018-12-14 2022-01-27 University Of Georgia Research Foundation, Inc. Crimean-congo hemorrhagic fever virus replicon particles and use thereof
US20220184158A1 (en) 2018-12-21 2022-06-16 Glaxosmithkline Biologicals Sa Methods of inducing an immune response
WO2020128893A1 (en) 2018-12-21 2020-06-25 Pfizer Inc. Combination treatments of cancer comprising a tlr agonist
GB201901608D0 (en) 2019-02-06 2019-03-27 Vib Vzw Vaccine adjuvant conjugates
JP2022525431A (en) 2019-04-02 2022-05-13 サノフイ Antigenic multirespiratory syncytial virus polypeptide
CA3136278A1 (en) 2019-04-10 2020-10-15 Pfizer Inc. Immunogenic compositions comprising conjugated capsular saccharide antigens, kits comprising the same and uses thereof
US20220221455A1 (en) 2019-04-18 2022-07-14 Glaxosmithkline Biologicals Sa Antigen binding proteins and assays
WO2020234300A1 (en) 2019-05-20 2020-11-26 Valneva Se A subunit vaccine for treatment or prevention of a respiratory tract infection
US20220249646A1 (en) 2019-05-25 2022-08-11 Infectious Disease Research Institute Composition and method for spray drying an adjuvant vaccine emulsion
WO2020240268A1 (en) 2019-05-31 2020-12-03 Universidad De Chile An immunogenic formulation that induces protection against shiga toxin-producing escherichia coli (stec)
EP3770269A1 (en) 2019-07-23 2021-01-27 GlaxoSmithKline Biologicals S.A. Quantification of bioconjugate glycosylation
CN114667158A (en) 2019-08-05 2022-06-24 葛兰素史克生物有限公司 Immunogenic compositions
EP4021894A1 (en) 2019-08-30 2022-07-06 University of Rochester Septin inhibitors for treatment of cancers
WO2021055580A2 (en) 2019-09-18 2021-03-25 Children's Medical Center Corporation An anaplastic lymphoma kinase (alk) cancer vaccine and methods of use
EP3799884A1 (en) 2019-10-01 2021-04-07 GlaxoSmithKline Biologicals S.A. Immunogenic compositions
KR20220092572A (en) 2019-11-01 2022-07-01 화이자 인코포레이티드 Escherichia coli composition and method therefor
EP4058581A1 (en) 2019-11-15 2022-09-21 Infectious Disease Research Institute Rig-i agonist and adjuvant formulation for tumor treatment
US20220387614A1 (en) 2019-11-22 2022-12-08 Glaxosmithkline Biologicals Sa Dosage and administration of a bacterial saccharide glycoconjugate vaccine
AU2020410410A1 (en) 2019-12-17 2022-06-09 Pfizer Inc. Antibodies specific for CD47, PD-L1, and uses thereof
WO2021127271A1 (en) 2019-12-17 2021-06-24 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Live attenuated leishmania parasite vaccines with enhanced safety characteristics
AU2021223184B2 (en) 2020-02-23 2024-10-10 Pfizer Inc. Escherichia coli compositions and methods thereof
EP4110382A1 (en) 2020-02-28 2023-01-04 Sanofi Pasteur Inc. High dose influenza vaccine for pediatric subjects
US11213482B1 (en) 2020-03-05 2022-01-04 University of Pittsburgh—Of the Commonwealth System of Higher Educat SARS-CoV-2 subunit vaccine and microneedle array delivery system
KR20230022246A (en) 2020-07-17 2023-02-14 화이자 인코포레이티드 Therapeutic Antibodies and Uses Thereof
JP7407081B2 (en) 2020-07-20 2023-12-28 三菱重工機械システム株式会社 Printing machines and how to clean up after them
WO2022051022A1 (en) 2020-09-04 2022-03-10 Infectious Disease Research Institute Co-lyophilized rna and nanostructured lipid carrier
WO2022066973A1 (en) 2020-09-24 2022-03-31 Fred Hutchinson Cancer Research Center Immunotherapy targeting pbk or oip5 antigens
CN116724053A (en) 2020-09-24 2023-09-08 弗雷德哈钦森癌症中心 Immunotherapy targeting SOX2 antigen
WO2022074061A1 (en) 2020-10-07 2022-04-14 Valneva Sweden Ab Cholera vaccine formulation
EP4234585A4 (en) 2020-10-23 2024-10-02 Jiangsu Provincial Center For Disease Control And Prevention Public Health Res Institute Of Jiangsu Fusion protein and application thereof
KR20230096033A (en) 2020-10-27 2023-06-29 화이자 인코포레이티드 Escherichia coli composition and method thereof
MX2023005221A (en) 2020-11-04 2023-05-16 Pfizer Immunogenic compositions for use in pneumococcal vaccines.
WO2022101745A2 (en) 2020-11-10 2022-05-19 Pfizer Inc. Immunogenic compositions comprising conjugated capsular saccharide antigens and uses thereof
KR20230148148A (en) 2020-12-23 2023-10-24 액세스 투 어드밴스드 헬스 인스티튜트 Solanesol vaccine adjuvant and method for preparing the same
US20220202923A1 (en) 2020-12-23 2022-06-30 Pfizer Inc. E. coli fimh mutants and uses thereof
WO2022147373A1 (en) 2020-12-31 2022-07-07 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Antibody-guided pcsk9-mimicking immunogens lacking 9-residue sequence overlap with human proteins
JP2024510717A (en) 2021-02-22 2024-03-11 グラクソスミスクライン バイオロジカルズ ソシエテ アノニム Immunogenic compositions, uses and methods
US20240156935A1 (en) 2021-03-31 2024-05-16 Vib Vzw Vaccine Compositions for Trypanosomatids
IL307519A (en) 2021-04-09 2023-12-01 Celldex Therapeutics Inc Antibodies against ilt4, bispecific anti-ilt4/pd-l1 antibody and uses thereof
EP4319802A2 (en) 2021-04-09 2024-02-14 Valneva SE Human metapneumovirus combination vaccine
CN117241813A (en) 2021-04-30 2023-12-15 卡利威尔免疫治疗公司 Oncolytic viruses for modified MHC expression
BR112023023671A2 (en) 2021-05-28 2024-02-06 Pfizer IMMUNOGENIC COMPOSITIONS COMPRISING CONJUGATED CAPSULAR SACHARIDE ANTIGENS AND USES THEREOF
WO2022249107A2 (en) 2021-05-28 2022-12-01 Pfizer Inc. Immunogenic compositions comprising conjugated capsular saccharide antigens and uses thereof
EP4370152A1 (en) 2021-07-16 2024-05-22 The Board Of Trustees Of The University Of Illinois Universal vaccine for influenza virus based on tetrameric m2 protein incorporated into nanodiscs
JP2024532763A (en) 2021-08-11 2024-09-10 サノフィ パスツール インコーポレイテッド Truncated influenza neuraminidase and methods of using same - Patents.com
IL311964A (en) 2021-10-08 2024-06-01 Sanofi Pasteur Inc Multivalent influenza vaccines
WO2023079507A1 (en) 2021-11-05 2023-05-11 Sanofi Respiratory syncytial virus rna vaccine
CA3237134A1 (en) 2021-11-05 2023-05-11 Timothy ALEFANTIS Multivalent influenza vaccines comprising recombinant hemagglutinin and neuraminidase and methods of using the same
EP4426345A1 (en) 2021-11-05 2024-09-11 Sanofi Hybrid multivalent influenza vaccines comprising hemagglutinin and neuraminidase and methods of using the same
WO2023077521A1 (en) 2021-11-08 2023-05-11 Celldex Therapeutics, Inc Anti-ilt4 and anti-pd-1 bispecific constructs
WO2023083964A1 (en) 2021-11-11 2023-05-19 2A Pharma Ab Parvovirus structural protein against beta- and gamma-hpv
CN118302191A (en) 2021-11-30 2024-07-05 圣诺菲·帕斯图尔公司 Human metapneumovirus vaccine based on viral vector
WO2023102373A1 (en) 2021-11-30 2023-06-08 Sanofi Pasteur Inc. Human metapneumovirus vaccines
WO2023114727A1 (en) 2021-12-13 2023-06-22 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Bacteriophage lambda-vaccine system
CA3242439A1 (en) 2021-12-17 2023-06-22 Sanofi Lyme disease rna vaccine
KR20240128715A (en) 2022-01-13 2024-08-26 화이자 인코포레이티드 Immunogenic composition comprising conjugated membrane saccharide antigen and use thereof
WO2023144206A1 (en) 2022-01-27 2023-08-03 Sanofi Pasteur Modified vero cells and methods of using the same for virus production
WO2023161817A1 (en) 2022-02-25 2023-08-31 Pfizer Inc. Methods for incorporating azido groups in bacterial capsular polysaccharides
WO2023177577A1 (en) 2022-03-14 2023-09-21 Sanofi Pasteur Inc. Machine-learning techniques in protein design for vaccine generation
WO2023214082A2 (en) 2022-05-06 2023-11-09 Sanofi Signal sequences for nucleic acid vaccines
WO2023218322A1 (en) 2022-05-11 2023-11-16 Pfizer Inc. Process for producing of vaccine formulations with preservatives
WO2023232901A1 (en) 2022-06-01 2023-12-07 Valneva Austria Gmbh Clostridium difficile vaccine
WO2024003239A1 (en) 2022-06-29 2024-01-04 Bavarian Nordic A/S RECOMBINANT MODIFIED saRNA (VRP) AND VACCINIA VIRUS ANKARA (MVA) PRIME-BOOST REGIMEN
WO2024052882A1 (en) 2022-09-09 2024-03-14 Access To Advanced Health Institute Immunogenic vaccine composition incorporating a saponin
WO2024094881A1 (en) 2022-11-04 2024-05-10 Sanofi Respiratory syncytial virus rna vaccination
WO2024110827A1 (en) 2022-11-21 2024-05-30 Pfizer Inc. Methods for preparing conjugated capsular saccharide antigens and uses thereof
US20240181028A1 (en) 2022-11-22 2024-06-06 Pfizer Inc. Immunogenic compositions comprising conjugated capsular saccharide antigens and uses thereof
WO2024116096A1 (en) 2022-12-01 2024-06-06 Pfizer Inc. Pneumococcal conjugate vaccine formulations
WO2024121380A1 (en) 2022-12-08 2024-06-13 Pierre Fabre Medicament Vaccinal composition and adjuvant
US20240189410A1 (en) 2022-12-13 2024-06-13 Pfizer Inc. Immunogenic compositions and methods for eliciting an immune response against clostridioides (clostridium) difficile
WO2024133515A1 (en) 2022-12-20 2024-06-27 Sanofi Rhinovirus mrna vaccine
WO2024149832A1 (en) 2023-01-12 2024-07-18 Bavarian Nordic A/S RECOMBINANT MODIFIED saRNA (VRP) FOR CANCER VACCINE
WO2024163327A1 (en) 2023-01-30 2024-08-08 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Epstein-barr virus glycoprotein 42 immunogens for vaccination and antibody discovery
WO2024166008A1 (en) 2023-02-10 2024-08-15 Pfizer Inc. Immunogenic compositions comprising conjugated capsular saccharide antigens and uses thereof
WO2024180262A1 (en) 2023-03-02 2024-09-06 Sanofi Compositions for use in treatment of chlamydia
WO2024186635A2 (en) 2023-03-03 2024-09-12 Celldex Therapeutics, Inc. Anti-stem cell factor (scf) and anti-thymic stromal lymphopoietin (tslp) antibodies and bispecific constructs
WO2024201324A2 (en) 2023-03-30 2024-10-03 Pfizer Inc. Immunogenic compositions comprising conjugated capsular saccharide antigens and uses thereof
WO2024214016A1 (en) 2023-04-14 2024-10-17 Pfizer Inc. Immunogenic compositions comprising conjugated capsular saccharide antigens and uses thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2034118A1 (en) † 1970-07-09 1972-01-13 Seinfeld, Hugo, Brendel, Walter, Prof Dr . 8000 München Xenogenic nucleic acids for enhancing antigenicity - of peptides and proteins
EP0468520A2 (en) † 1990-07-27 1992-01-29 MITSUI TOATSU CHEMICALS, Inc. Immunostimulatory remedies containing palindromic DNA sequences
WO1994002499A1 (en) † 1992-07-27 1994-02-03 Hybridon, Inc. Oligonucleotide alkylphosphonothioates

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4956296A (en) * 1987-06-19 1990-09-11 Genex Corporation Cloned streptococcal genes encoding protein G and their use to construct recombinant microorganisms to produce protein G
US5059519A (en) * 1986-07-01 1991-10-22 University Of Massachusetts Medical School Oligonucleotide probes for the determination of the proclivity for development of autoimmune diseases
CA1339596C (en) 1987-08-07 1997-12-23 New England Medical Center Hospitals, Inc. Viral expression inhibitors
US5786189A (en) * 1989-11-29 1998-07-28 Smithkline Beecham Biologicals (S.A.) Vaccine
US5248670A (en) 1990-02-26 1993-09-28 Isis Pharmaceuticals, Inc. Antisense oligonucleotides for inhibiting herpesviruses
JPH04352724A (en) * 1990-07-27 1992-12-07 Mitsui Toatsu Chem Inc Immunomodulation type therapeutic agent
DE69126710T2 (en) 1990-08-16 1998-01-15 Isis Pharmaceutical, Inc., Carlsbad, Calif. OLIGONUCLEOTIDES FOR MODULATING THE EFFECTS OF CYTOMEGALOVIRUS INFECTIONS
US5234811A (en) * 1991-09-27 1993-08-10 The Scripps Research Institute Assay for a new gaucher disease mutation
WO1993010138A1 (en) * 1991-11-18 1993-05-27 Tanox Biosystems, Inc. Anti-sense oligonucleotides for isotype-specific suppression of immunoglobulin production
US5585479A (en) * 1992-07-24 1996-12-17 The United States Of America As Represented By The Secretary Of The Navy Antisense oligonucleotides directed against human ELAM-I RNA
US5466705A (en) 1992-07-24 1995-11-14 Kumiai Chemical Industry Co., Ltd. Triazole derivatives pest controlling agent and pest control method
WO1994029461A1 (en) * 1993-06-11 1994-12-22 Commonwealth Scientific And Industrial Research Organisation Method for specific silencing of genes by dna methylation
WO1995026204A1 (en) 1994-03-25 1995-10-05 Isis Pharmaceuticals, Inc. Immune stimulation by phosphorothioate oligonucleotide analogs
ATE328890T1 (en) * 1994-07-15 2006-06-15 Univ Iowa Res Found IMMUNOMODULATORY OLIGONUCLEOTIDES
US5629052A (en) 1995-02-15 1997-05-13 The Procter & Gamble Company Method of applying a curable resin to a substrate for use in papermaking

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2034118A1 (en) † 1970-07-09 1972-01-13 Seinfeld, Hugo, Brendel, Walter, Prof Dr . 8000 München Xenogenic nucleic acids for enhancing antigenicity - of peptides and proteins
EP0468520A2 (en) † 1990-07-27 1992-01-29 MITSUI TOATSU CHEMICALS, Inc. Immunostimulatory remedies containing palindromic DNA sequences
WO1994002499A1 (en) † 1992-07-27 1994-02-03 Hybridon, Inc. Oligonucleotide alkylphosphonothioates

Non-Patent Citations (50)

* Cited by examiner, † Cited by third party
Title
ADRIAN P. BIRD: "CpG islands as gene markers in the vertebrate nucleus", TIG - DECEMBER 1987, vol. 3, no. 12, ELSEVIER PUBLICATIONS, CAMBRIDGE, pages 342 - 347 †
ANDREW W. HEATH: "Cytokines and the Rational Choice of Immunological Adjuvants", CANCER BIOTHERAPY - 1994, vol. 9, no. 1,, MARY ANN LIEBERT, INC., PUBLISHERS †
ANJU NOHRIA & ROBERT H. RUBIN: "Cytokines as potential vaccine adjuvants", BIOTHERAPY - 1994, no. 7, KLUWER ACADEMIC PUBLISHERS, pages 261 - 269 †
ANTHONY C. ALLISON: "Adjuvants and Immune Enhancement", INTERNATIONAL JOURNAL OF TECHNOLOGY ASSESSMENT IN HEALTH CARE, vol. 1994, no. 10/01, CAMBRIDGE UNIVERSITY PRESS, pages 107 - 120 †
ARTHUR M. KRIEG: "CpG Motifs in Bacterial DNA and Their Immune Effects", ANNU. REV. IMMUNOL. - 2002, no. 20, ANNUAL REVIEWS, pages 709 - 760 †
ARTHUR M. KRIEG: "Mechanisms and applications of immune stimulatory CpG oligodeoxynucleotides", BIOCHIMICA ET BIPHYSICA ACTA - 1999, no. 1489, ELSEVIER SCIENCE B.V., pages 107 - 116 †
BALLAS ET AL: "Induction of NK Activity in Murine and Human Cells by CpG Motifs in Oligodeoxynucleotides and Bacterial DNA", THE JOURNAL OF IMMUNOLOGY - 1996, no. 157, THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS, pages 1840 - 1845 †
BIOLOGICAL ABSTRACTS, no. 579O, Philadelphia, PA, US; VAN OJIK ET AL: "Phase I/II study with CpG 7909 as adjuvant to vaccination with MAGE-3 protein in patients with MAGE-3 positive tumors" †
BRANDA ET AL: "Amplification of antibody production by phosphorothioate oligodeoxynucleotides", J LAB CLIN MED - SEPTEMBER 1996, vol. 128, no. 3, MOSBY-YEAR BOOK, INC., pages 329 - 338 †
BRANDA ET AL: "IMMUNE STIMULATRION BY AN ANTISENSE OLIGOMER COMPLEMENTARY TO THE REV GENE OF HIV-1", BIOCHEMICAL PHARMACOLOGY - 1993, vol. 45, no. 10, pages 2037 - 2043 †
C.A. JANEWAY, JR. AND P. TRAVERS: "THE IMMUNE SYSTEM IN HEALTH AND DISEASE", IMMUNO BIOLOGY - THIRD EDITION †
COOPER ET AL: "Safety and immunogenicity of CPG 7909 injection as an adjuvant to Fluarix influenza vaccine", VACCINE - 2004, no. 22, ELSEVIER LTD., pages 3136 - 3143 †
DALPKE ET AL: "Phosphodiester CpG oligonucleotides as adjuvants: polyguanosine runs enhance cellular uptake and improve immunostimulative activity of phosphodiester CpG oligonucleotides in vitro and in vivo", IMMUNOLOGY - 2002, no. 106, BLACKWELL SCIENCE LTD, pages 102 - 112 †
DANIEL L.Y. LEONG AND JON A. RUDBACH: "Antigenic Competition Between an Endotoxic Adjuvant and a Protein Antigen", INFECTION AND IMMUNITY - 1971, vol. 3, no. 2, AMERICAN SOCIETY FOR MICROBIOLOGY, pages 308 - 317 †
DAVID S. PISETSKY & CHARLES REICH: "Stimulation of in vitro proliferation of murine lymphocytes by synthetic oligodeoxynucleotides", MOLECULAR BIOLOGY REPORTS - 1993, vol. 18, KLUWER ACADEMIC PUBLISHERS, pages 217 - 221 †
DAVID S. PISETSKY AND CHARLES F. REICH: "STIMULATION OF MURINE LYMPHOCYTE PROLIFERATION BY A PHOSPHOROTHIOATE OLIGO- NUCLEOTIDE WITH ANTISENSE ACTIVITY FOR HERPES SIMPLEX VIRUS", LIFE SCIENCES 1993, vol. 54, PERGAMON PRESS LTD, pages 101 - 107 †
Declaration of Dr. George Weiner †
DENNIS M. KLINMAN: "IMMUNOTHERAPEUTIC USES OF CpG OLIGODEOXYNUCLEOTIDES", NATURE REVIEWS IMMUNOLOGY 2004, vol. 4, no. APRIL, NATURE PUBLISHING GROUP †
EUGEN UHLMANN & GOERG VOLLMER: "Recent advances in the development of immunostimulatory oligonucleotides", CURRENT OPINION IN DRUG DISCOVERY & DEVELOPMENT 2003, vol. 6 (2), CURRENT DRUGS, pages 204 - 217 †
FRANCOIS ET AL: "Examination of the Inhibitory and Stimulatory Effects of IFN-?, -?, and -? on Human B-Cell Proliferation Induced by Various B-Cell Mitogens", CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY - 1988, no. 48, ACADEMIC PRESS. INC., pages 297 - 306 †
GILKESON ET AL: "INDUCTION OF ANTI-DOUBLE STRANDED DNA ANTIBODIES IN NORMAL MICE BY IMMUNIZATION WITH BACTERIAL DNA", THE JOURNAL OF IMMUNOLOGY - 1989, vol. 142, no. 4, THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS, pages 1482 - 1486 †
GOECKERITZ ET AL: "Multivalent cross-linking of membrance lg sensitizes murine B cells to a broaderspectrum of CpG-containing oligodeoxynucleotide motifs, including their methylated counterparts, for stimulation of proliferation and lg secretion", INTERNATIONAL IMMUNOLOGY - 1999, vol. 11, no. 10, pages 1693 - 1700 †
HALPERIN ET AL: "A phase I study of the safety and immunogenicity of recombinant hepatitis B surface antigen co-administered with an immunostimulatory phosphorothioate oligonucleotide adjuvant", VACCINE - 2003, no. 21, ELSEVIER SCIENCE LTD., pages 2461 - 2467 †
ICHIRO AZUMA: "Synthetic immunoadjuvants: application to non-specific host stimulation and potentiation of vaccine immunogenicity", VACCINE - 1992, vol. 10, no. 14, BUTTERWORTH-HEINEMANN LTD †
IHO ET AL: "Oligodeoxynucleotides Containing Palindrome Sequences with Internal 5'-CpG-3' Act Directly on Human NK and Activated T Cells to Induce IFN-? Production In Vitro", THE JOURNAL OF IMMUNOLOGY - 1999, no. 163, THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS, pages 3642 - 3652 †
IVAN ROITT: "Textbook", ESSENTIAL IMMUNOLOGY - 1991, 7TH EDITION, BLACKWELL SCIENTIFIC PUBLICATIONS, pages 237 †
JANIS KUBY: "Textbook Immunology", LIBR. OF CONGRESS CATALOGING-IN-PUBLICATION DATA, CHAPTER 4, EPITOPES, vol. 1992, W.H. FREEMAN AND COMPANY, pages 78 - 79 †
KATAOKA ET AL: "Antitumor Activity of Synthetic Oligonucleotides with Sequences from cDNA Encoding Proteins of Mycobacterium bovis BCG", RAPID COMMUNICATION - MARCH 1992, no. 83, pages 244 - 247, XP002085539 †
KATHARINE MERRITT AND ARTHUR G. JOHNSON: "STUDIES ON THE ADJUVANT ACTION OF BACTERIAL ENDOTOXINS ON ANTIBODY FORMATION", THE JOURNAL OF IMMUNOLOGY - 1965, vol. 94, no. 3, THE WILLIEAMS & WILKINS CO., pages 416 - 422 †
KLINMAN ET AL: "CpG oligonucleotides improve the protective immune response induced by the anthrax vaccination of rhesus macaques", VACCINE, vol. 22, no. 2004, ELSEVIER LTD., pages 2881 - 2886 †
KRIEG ET AL: "Enhancing vaccines with immune stimulatory CpG DNA", vol. 3(1), no. 2001, PHARMAPRESS LTD †
KURAMOTO ET AL: "Induction of T-cell-mediated immunity against MethA fibrosarcoma by intratumoral injections of a bacillus Calmette-Guérin nucleic acid fraction", CANCER IMMUNOL IMMUNOTHER - 1992, no. 34, pages 283 - 288, XP008038287 †
KURAMOTO ET AL: "Oligonucleotide Sequences Rquired for Natural Killer Cell Activation", RAPID COMMUNICATION - NOVEMBER 1992, vol. 83, pages 1128 - 1131 †
LEE ET AL: "Effects of a Hexameric Deoxyriboguanosine Run Conjugation into CpG Oligodeoxynucleotides on Tehir Immunostimulatory Potentials", THE JOURNAL OF IMMUNOLOGY - 2000, no. 165, THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS, pages 3631 - 3639 †
LIU ET AL: "Immunostimulatory CpG Oligodeoxynucleotides Enhance the Immune Response to Vaccine Strategies Involving Granulocyte-Macrophage Colony-Stimulating Factor", BLOOD 1998, vol. 92, no. 10, pages 3730 - 3736, XP002125531 †
LIVINGSTON ET AL: "SEROLOGICAL RESPONSE OF MELANOMA PATIENTS RECEIVING MELANOMA CELL VACCINES. I. AUTOLOGOUS CULTURED MELANOMA CELLS", INT. J. CANCER - 1982, no. 30, pages 413 - 422 †
LOTZ ET AL: "Effect of Recombinant Human Interferons on Rheumatotoid Arthritis B Lymphocytes Activated by Epstein-Barr Virus", THE JOURNAL OF RHEUMATOLOGY 1987, no. 14, pages 42 - 45 †
MCINTYRE ET AL: "A Sencse Phosphorothioate Oligonucleotide Directed to the Initiation Codon of Transcription Factor NF-kB p65 Causes Sequence-Specific Immune Stimulation", ANTISENSE RESEARCH AND DEVELOPMENT - 1993, vol. 3, MARY ANN LIEBERT, INC., PUBLISHERS, pages 309 - 322 †
MESSINA ET AL: "STIMULATION OF IN VITRO MURINE LYMPHOCYTE PROLIFERATION BY BACTERIAL DNA", THE JOURNAL OF IMMUNOLOGY - SEPTEMBER 1991, vol. 147, no. 6, pages 1759 - 1764 †
MESSINA ET AL: "The Influence of DNA Structure on the in Vitro Stimulation of Murine Lymphocytes by Natural and Synthetic Polynucleotide Antigens", CELLULAR IMMUNOLOGY - 1993, no. 147, ACADEMIC PRESS. INC., pages 148 - 157 †
MOJCIK ET AL: "Administration of a Phosphorothiate Oligonucleotide Antisense to Murine Endogenous Retroviral MCF env. Causes Immune Effects in Vivo in a Sequence-Specific", CLINICAL IMMUNOLGOY AND IMMUNOPATHOLOGY - 1993, no. 67, pages 130 - 136 †
SAIKI ET AL: "Induction of tumoricidal macrophages and production of cytokines by synthetic muramyl dipeptide analogues", VACCINE - 1988, vol. 6, BUTTERWORTH & CO., pages 238 - 244 †
SCOTT F. GILBERT, DEVELOPMENTAL BIOLOGY - THIRD EDITION, vol. 1991, SINAUER ASSOCIATES, INC., pages 432 - 436 †
VERTHELYI ET AL: "CpG oligodeoxynucleotides improve the response to hepatitis B immunization in healthy and SIV-infected rhesus macaques", CONCISE COMMUNICATION - AIDS 2004, no. 18, LIPPINCOTT WILLIAMS & WILKINS, pages 1003 - 1008 †
VERTHELYI ET AL: "Human Peripheral Blood Cells Differentially Recognize and Respond to Two Distinct CpG Motifs", THE JOURNAL OF IMMUNOLOGY - 2001, no. 166, THE AMERICAN ASSOCIATION OF IMMUNOLGISTS, pages 2372 - 2377 †
WEINER ET AL: "Immunostimulatory oligodeoxynucleotides containing the CpG motif are effective as immune adjuvants in tumor antigen immunization", PROC. NATL. ACAD. SCI - 1997, vol. 94, THE NATIONAL ACADEMY OF SCIENCES, pages 10833 - 10837 †
WHEATER'S FUNCTIONAL HISTOLOGY - 3RD EDITION, no. 1993 †
YAMAMOTO ET AL: "Ability of Oligonucleotides with Certain Palindromes to Induce Interferon Production and Augment Natural Killer Cell Activity Is Associated with their Base Length", ANTISENSE RESEARCH AND DEVELOPMENT - 1994, no. 4, MARY ANN LIEBERT, INC. PUBLISHERS, pages 119 - 122 †
YAMAMOTO ET AL: "Lipofection of Synthetic Oligodeoxyribonucleotide Having a Palindromic Sequence of AACGTT to Murine Splenocytes Enhances Interferon Production and Natural Killer Activity", MICROBIOL. IMMUNOL., vol. 1994, no. 38, pages 831 - 836 †
YU ET AL: "Potent CpG oligonucleotides containing phosphodiester linkages: in vitro and in vivo immunostimulatory properties", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS - 2002, no. 297, ELSEVIER SCIENCE (USA), pages 83 - 90 †

Also Published As

Publication number Publication date
ES2267100T3 (en) 2007-03-01
JPH10506265A (en) 1998-06-23
ES2320315T5 (en) 2012-12-05
DE69535036T3 (en) 2011-07-07
JP2008169216A (en) 2008-07-24
JP3468773B2 (en) 2003-11-17
ES2320315T3 (en) 2009-05-21
JP2003144184A (en) 2003-05-20
WO1996002555A1 (en) 1996-02-01
EP1167377A3 (en) 2004-09-08
JP2004215670A (en) 2004-08-05
HK1044950A1 (en) 2002-11-08
EP1167378A3 (en) 2005-08-17
ATE420171T1 (en) 2009-01-15
EP1167379A2 (en) 2002-01-02
DK0772619T3 (en) 2006-10-09
HK1044949A1 (en) 2002-11-08
EP1167379A3 (en) 2004-09-08
JP2004043466A (en) 2004-02-12
EP0772619B1 (en) 2006-06-07
EP1167377B1 (en) 2009-01-07
PT772619E (en) 2006-10-31
ATE509102T1 (en) 2011-05-15
ES2366201T3 (en) 2011-10-18
ATE328890T1 (en) 2006-06-15
ES2267100T5 (en) 2011-04-08
DE69535036T2 (en) 2007-01-18
EP1167378B1 (en) 2011-05-11
EP1167378A2 (en) 2002-01-02
AU713040B2 (en) 1999-11-18
DE69535905D1 (en) 2009-02-26
DK0772619T4 (en) 2011-02-21
EP0772619A1 (en) 1997-05-14
DE69535036D1 (en) 2006-07-20
CA2194761C (en) 2006-12-19
CA2194761A1 (en) 1996-02-01
EP1167377B2 (en) 2012-08-08
EP1167377A2 (en) 2002-01-02
HK1043598A1 (en) 2002-09-20
CA2560114A1 (en) 1996-02-01
HK1044949B (en) 2012-01-27
JP4126252B2 (en) 2008-07-30
US6194388B1 (en) 2001-02-27
JP2004024261A (en) 2004-01-29
AU1912795A (en) 1996-02-16
JP2009148296A (en) 2009-07-09

Similar Documents

Publication Publication Date Title
EP0772619B2 (en) Immunomodulatory oligonucleotides
US8309527B2 (en) Immunomodulatory oligonucleotides
AU754463B2 (en) Immunomodulatory oligonucleotides

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19970213

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL PT SE

17Q First examination report despatched

Effective date: 19970506

TPAD Observations filed by third parties

Free format text: ORIGINAL CODE: EPIDOS TIPA

RIN1 Information on inventor provided before grant (corrected)

Inventor name: STEINBERG, ALFRED D.

Inventor name: KLINMAN, DENNIS

Inventor name: KRIEG, ARTHUR, M.

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: COLEY PHARMACEUTICAL GROUP, INC.

Owner name: UNITED STATES OF AMERICA, AS REPRESENTED BY THE SE

Owner name: THE UNIVERSITY OF IOWA RESEARCH FOUNDATION

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL PT SE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT;WARNING: LAPSES OF ITALIAN PATENTS WITH EFFECTIVE DATE BEFORE 2007 MAY HAVE OCCURRED AT ANY TIME BEFORE 2007. THE CORRECT EFFECTIVE DATE MAY BE DIFFERENT FROM THE ONE RECORDED.

Effective date: 20060607

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REF Corresponds to:

Ref document number: 69535036

Country of ref document: DE

Date of ref document: 20060720

Kind code of ref document: P

REG Reference to a national code

Ref country code: SE

Ref legal event code: TRGR

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: MOINAS & SAVOYE SA

REG Reference to a national code

Ref country code: GR

Ref legal event code: EP

Ref document number: 20060402943

Country of ref document: GR

REG Reference to a national code

Ref country code: DK

Ref legal event code: T3

REG Reference to a national code

Ref country code: PT

Ref legal event code: SC4A

Effective date: 20060905

ET Fr: translation filed
REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2267100

Country of ref document: ES

Kind code of ref document: T3

PLBI Opposition filed

Free format text: ORIGINAL CODE: 0009260

PLAX Notice of opposition and request to file observation + time limit sent

Free format text: ORIGINAL CODE: EPIDOSNOBS2

26 Opposition filed

Opponent name: CYTOS BIOTECHNOLOGY AG

Effective date: 20070307

Opponent name: MOLOGEN AG

Effective date: 20070306

Opponent name: DYNAVAX TECHNOLOGIES CORPORATION

Effective date: 20070306

NLR1 Nl: opposition has been filed with the epo

Opponent name: CYTOS BIOTECHNOLOGY AG

Opponent name: MOLOGEN AG

Opponent name: DYNAVAX TECHNOLOGIES CORPORATION

PLAF Information modified related to communication of a notice of opposition and request to file observations + time limit

Free format text: ORIGINAL CODE: EPIDOSCOBS2

PLAF Information modified related to communication of a notice of opposition and request to file observations + time limit

Free format text: ORIGINAL CODE: EPIDOSCOBS2

PLBP Opposition withdrawn

Free format text: ORIGINAL CODE: 0009264

PLAF Information modified related to communication of a notice of opposition and request to file observations + time limit

Free format text: ORIGINAL CODE: EPIDOSCOBS2

PLBB Reply of patent proprietor to notice(s) of opposition received

Free format text: ORIGINAL CODE: EPIDOSNOBS3

PLAB Opposition data, opponent's data or that of the opponent's representative modified

Free format text: ORIGINAL CODE: 0009299OPPO

PLBP Opposition withdrawn

Free format text: ORIGINAL CODE: 0009264

PLAB Opposition data, opponent's data or that of the opponent's representative modified

Free format text: ORIGINAL CODE: 0009299OPPO

APBM Appeal reference recorded

Free format text: ORIGINAL CODE: EPIDOSNREFNO

APBP Date of receipt of notice of appeal recorded

Free format text: ORIGINAL CODE: EPIDOSNNOA2O

APAH Appeal reference modified

Free format text: ORIGINAL CODE: EPIDOSCREFNO

APBU Appeal procedure closed

Free format text: ORIGINAL CODE: EPIDOSNNOA9O

PUAH Patent maintained in amended form

Free format text: ORIGINAL CODE: 0009272

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: PATENT MAINTAINED AS AMENDED

27A Patent maintained in amended form

Effective date: 20101208

AK Designated contracting states

Kind code of ref document: B2

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL PT SE

REG Reference to a national code

Ref country code: CH

Ref legal event code: AEN

Free format text: BREVET MAINTENU DANS UNE FORME MODIFIEE

REG Reference to a national code

Ref country code: NL

Ref legal event code: T3

REG Reference to a national code

Ref country code: DK

Ref legal event code: T4

REG Reference to a national code

Ref country code: PT

Ref legal event code: MP4A

Effective date: 20110309

REG Reference to a national code

Ref country code: SE

Ref legal event code: RPEO

REG Reference to a national code

Ref country code: ES

Ref legal event code: DC2A

Ref document number: 2267100

Country of ref document: ES

Kind code of ref document: T5

Effective date: 20110408

REG Reference to a national code

Ref country code: GR

Ref legal event code: EP

Ref document number: 20110400322

Country of ref document: GR

Effective date: 20110317

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20140227

Year of fee payment: 20

Ref country code: SE

Payment date: 20140227

Year of fee payment: 20

Ref country code: LU

Payment date: 20140305

Year of fee payment: 20

Ref country code: IE

Payment date: 20140227

Year of fee payment: 20

Ref country code: MC

Payment date: 20140120

Year of fee payment: 20

Ref country code: CH

Payment date: 20140227

Year of fee payment: 20

Ref country code: NL

Payment date: 20140226

Year of fee payment: 20

Ref country code: DK

Payment date: 20140225

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: IT

Payment date: 20140225

Year of fee payment: 20

Ref country code: FR

Payment date: 20140220

Year of fee payment: 20

Ref country code: ES

Payment date: 20140226

Year of fee payment: 20

Ref country code: GR

Payment date: 20140227

Year of fee payment: 20

Ref country code: AT

Payment date: 20140121

Year of fee payment: 20

Ref country code: BE

Payment date: 20140228

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: PT

Payment date: 20140124

Year of fee payment: 20

Ref country code: GB

Payment date: 20140227

Year of fee payment: 20

REG Reference to a national code

Ref country code: DE

Ref legal event code: R071

Ref document number: 69535036

Country of ref document: DE

REG Reference to a national code

Ref country code: DK

Ref legal event code: EUP

Effective date: 20150207

REG Reference to a national code

Ref country code: DE

Ref legal event code: R071

Ref document number: 69535036

Country of ref document: DE

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: PT

Ref legal event code: MM4A

Free format text: MAXIMUM VALIDITY LIMIT REACHED

Effective date: 20150207

REG Reference to a national code

Ref country code: NL

Ref legal event code: V4

Effective date: 20150207

REG Reference to a national code

Ref country code: GB

Ref legal event code: PE20

Expiry date: 20150206

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK07

Ref document number: 328890

Country of ref document: AT

Kind code of ref document: T

Effective date: 20150207

REG Reference to a national code

Ref country code: IE

Ref legal event code: MK9A

REG Reference to a national code

Ref country code: SE

Ref legal event code: EUG

REG Reference to a national code

Ref country code: GR

Ref legal event code: MA

Ref document number: 20110400322

Country of ref document: GR

Effective date: 20150208

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION

Effective date: 20150207

Ref country code: PT

Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION

Effective date: 20150216

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION

Effective date: 20150206

REG Reference to a national code

Ref country code: ES

Ref legal event code: FD2A

Effective date: 20150826

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION

Effective date: 20150208